CN105899228A - Live attenuated vaccines - Google Patents
Live attenuated vaccines Download PDFInfo
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- CN105899228A CN105899228A CN201480067569.8A CN201480067569A CN105899228A CN 105899228 A CN105899228 A CN 105899228A CN 201480067569 A CN201480067569 A CN 201480067569A CN 105899228 A CN105899228 A CN 105899228A
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Abstract
The present invention refers to a method for the production of live attenuated bacterial strains, suitable as vaccine candidates, comprising the steps of: A. providing a bacterial strain capable of expressing glutamate racemase and possibly D-amino acid transaminase and comprising a peptidoglycan cell wail, and B. inactivating the gene or genes encoding for the glutamate racemase enzyme and, if needed, the gene or genes encoding for the enzyme D-amino acid transaminase in such way that the bacterial strain is no longer capable of expressing a functional glutamate racemase and/or a functional D-amino acid transaminase; wherein the inactivation of said genes causes said bacterial strain to be auxotrophic for D- glutamate.
Description
Technical field
The present invention provides living toxic-reduced bacteria vaccine.The method that the present invention also provides for obtaining this kind of vaccine.
Background technology
Homoiothermic animal including people overcomes the pathogenetic mode of microorganism to be a complicated process.To microorganism
Pathogenetic immunity is that homoiothermic animal is avoided pathogenesis or suffers a kind of mode of less fierce pathogenic state.To giving
Determine morbidity and death that the incomplete immunity of pathogen causes being exposed in the colony of pathogen.It is generally acknowledged based on living still
The immunne response of vaccine (attenuated live vaccine) the inducement efficient type of the microorganism of attenuation.This kind of vaccine has advantage, once moves
Thing host immunity inoculation, microbial pathogens enters that host can induce acceleration to recall earlier, cell-mediated or body fluid is exempted from
Epidemic disease, its can infect can present notable ratio clinically before control the further growth of organism.Typically recognize based on
The vaccine (inactivated vaccine) of the pathogen killed can not obtain such response.But, according to attenuation, comprise disease of living
The vaccine of substance is dangerous, when immunity inoculation, and the disease that host's PI of immunity inoculation seeks protection.Therefore, tool
The immune attribute having viable microbial still can not cause the vaccine of adverse side effect to be desirable when immunity inoculation.
It is important to note however that attenuated bacteria bacterial strain cannot be only by so as effectively using of vaccine candidate object
Attenuation predict.In this respect, the conventional method weakening antibacterial is to remove one or more virulence factor (genetic modifications
Organism GMO), but, in most of the cases, virulence factor also plays a role as protective epitope in induction immunity.
Under those circumstances, the disappearance of virulence factor inevitably weakens the immunogenic abilities of antibacterial.That yes is undesirable for this
Situation.Therefore, live vaccine should preferably retain the antigen complement of wild-type strain.
Additionally, once set up attenuation, to the immunne response of certain types of vaccine candidate object and comprise this kind of micro-
The success of biological vaccine combination nevertheless suffers from being permitted as will be detailed later multifactorial impact:
A. attenuated live vaccine bacterial strain preferably substantially should can not reply its initial condition (usually virulent wt bacterium
Strain), and other genes should not supplement operated gene, cause antibacterial can cause disease (preferably stable sudden change).
B. discounting for, in live vaccine, endotoxic existence can be shortcoming, because these molecules can cause seriously
General reaction.And, the administration of whole-cell vaccines is local response originality (severe pain, local swelling and edema, panniculitis
Or ulcer etc.) classical risk factor.
C. the vigor and the fitness that are attenuated GMO should be by dramatic impact, since it is desirable that occur some to replicate in vivo
To produce enough microorganisms and antigen thereof with stimulating immune system.It is true that any sudden change in gene all may interfere with multiple
System maybe may damage the viable microbial in bottle, causes vaccine failure.Therefore, it is necessary to carefully evaluate each type of genetic modification pair
The unexpectedness effect of cell.
D. additionally, when select gene target for genetic manipulation time, it should consider gene share or the part-time thus egg of albumen
The phenomenon of more than one function can be performed in vain.Many part-time enzymes of albumen.One example is glutamate racemase (MurI), and it is
Key enzyme in cell wall synthesis, but also work in gyrase suppresses.Due to its multi-functional feature, it is not possible to pre-
Survey the availability of targeting MurI strategy, unless the sudden change obtained in these genes evaluate impact physiological on bacterial cell.
E. additionally, the type of the immunne response of vaccine initiation may be not suitable for providing enough protections (the vaccine mistake to infecting
Lose).Specific requirement to effective vaccine can change according to the character of pathogen.In the case of extracellular pathogen, mainly
The defence that antibody is organism provides adaptation mechanism, and the existence of T cell is essential in controlling intracellular organisms.Knot
Really, attenuated live vaccine is used as preferable immunogen, and it kills antibacterial by simple operations or subunit forms, and modifies internal sense
The bacterial antigens occurred during dye.Therefore, Attenuated strain can cause more extensive and sufficient immunne response, particularly carefully
The intracellular stage.In this sense, the target gene strategy of attenuation carefully should be tested in vaccine candidate object, because operated
Antibacterial utilize the ability of the natural route infected may be impaired and do not trigger the immunne response of extensively protection.
F. additionally, unrelated attenuation or the type of immune response of initiation, it is administered with specific GMO to reach acceptable guarantor
The dose quantity (effectively with persistently) protecting level can continue for vaccine timetable.
G. additionally, the route of administration of vaccine may decide that the type of immune response of increase, and for its success to closing weight
Want.According to route of administration, vaccine can enter organism by different way: (in this case, antigen is drenched skin by region
The Langerhans cell serving as antigen-presenting cell in the T-district fawned on absorbs);(capture of antigen is mainly thin by M here for mucosa
Born of the same parents are carried out, and immunne response develops in aggregate nodules) or blood (antigen can targeting spleen, here it can bite by spleen is huge
Cell is processed).As a result, GMO once setting up attenuation, the position that vaccine is administered may decide that failure or the one-tenth of immunity inoculation
Merit.In this respect, have proven in breast rather than intraperitoneal administration attenuated live staphylococcus aureus (S.aureus) bacterial strain shows
Write the bacterial load in mammary gland after minimizing wild-type strain is attacked.The vaccine candidate object staphylococcus aureus proposed
8325-4 A523 is the temperature sensitive mutant separated after nitrosoguanidine mutagenesis, and it replicates under low temperature (less than 32 DEG C)
Well, but when being transferred to mammal body temperature experience limited quantity division.Author uses staphylococcus aureus 8325-4
Wild-type strain carries out attacking experiment and makees with the bacterial load in mammary gland between relative immunity inoculation and the animal of non-immunity inoculation
Tolerance for vaccine Vaccine effectiveness.These authors are concluded that and " recover from by the mammary gland of mouse of intramammary route immunity
Staphylococcus aureus CFU quantity be substantially less than (7x102CFU) the staphylococcus aureus CFU found in control mice
Quantity (1.5x105CFU).On the contrary, from by any intraperitoneal scheme immunity mammary gland of mouse recover CFU quantity with from right
The CFU quantity the highest (P > 0.5) recovered according to mice gland ".Therefore, i.e. using potential material standed for, route of administration still can be determined
Determine the attenuated live mutant effect as vaccine.
H. last, vaccine can not induce the cross reacting antibody of the multiple bacterial strains for identical bacterial species.Although
Attenuated strain can cause the antibody of protection in animal model, but is normally only being used for producing the parent bacterium of vaccine strains
Strain is also used for during Attack Research seeing this protection.The most reliable generation of broad-based protection or survey for other bacterial strains
Examination.Although additionally, fully cause and cross reaction, but produced antibody can in the model with wild type following pathogen challenge
Can not last for a long time and unprotected property.
Generally speaking, live vaccine should enough be attenuated (or nontoxic) to avoid unacceptable pathologic effect, but separately
On the one hand, it must cause enough immunne response, described immunne response can give independent of bacterial isolates in host
Persistently protection (protective immunity) for disease.
Confirm live vaccine be enough attenuation (or nontoxic) with avoid unacceptable pathologic effect and initiation can be host
Middle imparting is not easy independent of the enough immunne response for the persistently protection (protective immunity) of disease of bacterial isolates
Task.In this sense, WO99/25376 describe a kind of cause in mammal for antigen T cell immunity should
The method answered, described method includes the nutrition being administered the Listerella (Listeria) expressing described antigen to described mammal
Deficiency attenuated strain.Wherein said auxotroph attenuated strain is described as having sudden change at least one gene, described
The protein product of gene is essential for listerial growth.Especially, the present invention describes the battalion of D-alanine synthesis
Supporting deficiency attenuated strain, it comprises the DNA of encoding heterologous antigen further, and wherein said heterologous antigen is preferably HIV-1 and resists
Former.
In WO99/25376, result is expressed as showing that listerial auxotrophic strain carries in BALB/c mouse
For the protection attacked for Listeria monocytogenes (L.monocytogenes) so that this bacterial strain is it is said that be suitable for vaccine group
Compound, the protection of the infection for causing for this organism.But, the experiment embodiment wherein provided only sets up single increasing Lee
The attenuation auxotroph D-alanine mutant of this special bacterium causes CTL (host cell toxic T cell) response.Wherein do not consider
Antibody-mediated immunne response (humoral immunization), and result is not provided in this sense.Additionally, it is wherein wild by measurement
After the attack of raw type Listerella, the count of bacteria in the spleen of infecting mouse determines the protected effect of this mutant.At this
In meaning, only just can evaluate vaccine when carrying out survival and measuring and (particularly cause for acute and lethal bacteria infection
Those of septicopyemia) effectiveness.Additionally, the mutant Listerella without D-alanine injection is described as providing seldom guarantor
Protect.By contrast, when supplementing D-alanine in the initial inoculation thing of mutation biology body to reach identical with wild-type strain
During protection (when primary immune is inoculated), this has the effect of the fatal dose about 10 times reducing mutant, if it is considered that attenuation
Loss this be the serious restriction of safety of this mutant.Therefore, WO99/25376 is the D-alanine described in it
The result that mutant provides can not confirm that these mutants which hads are as sexy for extracellular bacteria pathogen and acute systemic
The availability of the vaccine candidate object of dye.Cannot ensure by D-alanine additionally, lack the cross protection data with these mutants
The vaccine of auxotroph composition produces the effectiveness of the extensive protective immune response for other Listeria monocytogenes bacterial strains,
Not to mention it is for producing the availability of the vaccine candidate object of other bacterial species.
Additionally, in the detailed description of WO99/25376, inventor proposes suggested below: " the volume for extra generation
Outer potentially useful target includes the gene participating in the synthesis of cell-wall component D-Glu.Lack to produce D-Glu nutrition
Swaged mutant, it is necessary to inactivation dat gene, dat gene participates in D-glu+pyr and is converted to α-ketoglutaric acid+D-ala and converse
Should.Also must inactivate glutamate racemase gene dga ".But, one skilled in the art will recognize that according to the information that there is shown
There is no rational chance of success: listerial D-Glu auxotrophic strain may give gratifying attenuation
To avoid unacceptable pathologic effect and to cause the enough immunity that can give the persistently protection for disease in host to answer
Answer (protective immunity).As discussed above, each gene target strategy should be evaluated one by one.Additionally, glutamate racemase tool
There is Moonlighting functions, if operating its encoding gene, cell survival can be affected.In this sense, from before invention not
May infer that glutamate racemase as target with produce D-Glu auxotrophic vaccines bacterial strain to give directed toward bacteria sense
The purposes of the protection of dye, because not continuing evidence, and such bacterial strain can be immunogenic and inconspicuous.
This explanation of the latter, the most such D-Glu auxotrophic vaccines bacterial strain can be immunogenic and fail to understand
Aobvious, by the fact that support further, under current vaccine development state, research or invention do not confirm D-Glu battalion
Support deficiency microorganism and can be used as the ability that live vaccine gives the protection of the disease that directed toward bacteria causes.In this sense, and
Although and the 23rd ESCMID conference (clinical microbe of europe and the lemology held in Berlin 27-30 day in April, 2013
Meeting) summary M.P.Cabral et al, " Blockade of glutamate racemisation during cell-
wall formation prevents biofilm and proliferation of Acinetobacter baumannii
In vivo " in confirm D-Glu Auxotrophic mutant sizable attenuation (display D-Glu nutrition lack
The unique reference document of the relation between swaged and internal forfeiture virulence), it is noted that one of major obstacle of vaccine development
It it is the immunogenicity (protection) being difficult to reach gratifying attenuation and seriously damaging.Accordingly, it would be desirable to each base
Because targeting amendment strategy always tests the effective vaccine that the relation between attenuation with protection infects with exploitation directed toward bacteria.At this
In meaning, ((European clinical is micro-in the 23rd the ESCMID conference held in Berlin 27-30 day in April, 2013 for file mentioned above
Biology and infectious disease association) summary M.P.Cabral et al, " Blockade of glutamate racemisation
during cell-wall formation prevents biofilm and proliferation of
Acinetobacter baumannii in vivo "), even if which depict in the frame being characterised by glutamate racemase gene
The attenuation of Acinetobacter bauamnnii (Acinetobacter baumannii) bacterial strain of disappearance, but it is not provided that any data show
Show the Vaccine effectiveness that such bacterial strain infects for Acinetobacter bauamnnii (A.baumannii).In following prior art document
Confirm, it is provided that the Vaccine effectiveness of data display attenuated strain is for determining that such bacterial strain availability as vaccine is to closing weight
Want.
At M.K.Hondalus et al, " Attenuation of and protection induced by a
leucine auxotroph of Mycobacterium tuberculosis”,Infection and Immunity 68
(2000), in 2888-2898, the leucine nutrition being produced mycobacterium tuberculosis (M.tuberculosis) by equipotential exchange is lacked
Swaged, thus mutant can not replicate (confirming that antibacterial is attenuation) in macrophage.It is true that Fig. 5 of this file shows
Show how the mice of the leucine auxotroph infecting mycobacterium tuberculosis has 100% survival rate (card after infection for 22 weeks
Being attenuated really of real leucine auxotrophy type).But, reducing intravenous attack according to display leucine auxotroph mutant
BALB/c mouse organ burden and histopathology aspect not as good as live BCG vaccine effective.The explanation of this file has attenuation
Bacterial strain is not sufficient to have vaccine, and immunogenicity is key issue.
Additionally, at M.S.Jr Pavelka et al, " Vaccine efficacy of a lysisne auxotroph
Of Mycobacterium tuberculosis ", in Infection and Immunity 71 (2003) 4190-4192, according to
In confirmation mycobacterium tuberculosis mutant (the lysine auxotroph of mycobacterium tuberculosis) azygos vein, immunity inoculation is little
Mus does not produce aerocolloidal notable protective response subsequently, and relative to by the single immunity inoculation of BCG, uses auxotrophy
The single immunity inoculation of type is not enough to reduce the bacterial load in lung and spleen.Only three immunity mouse survivals with use single dose
The control mice of BCG immunity inoculation is the longest.As a result, again it may be concluded that immunogenicity be from attenuation key and
Individually problem.
Additionally, prior art references Ann-Mari Svennerholm et al, " Vaccines against
Enterotoxigenic Escherichia coli ", Expert review of vaccines 7 (2008) 795-804 retouches
Stated ETEC (enterotoxigenic escherichia coil (Escherichia the coli)) bacterial strain of hereditary attenuation as live oral carrier and
It is characterized as safety.But when evaluating these identical bacterial strains and being used for protecting, the sickness rate of diarrhoea in vaccine is to placebo receptor
Or always feces amount does not all substantially reduce.
Finally, H.K.Kim et al, " Identifying protective antigens of Staphylococcus
aureus,a pathogen that suppresses host immune responses”,FASEB J.25(2011)
Whether 3605-3612 describes can be little derived from 3 kinds of attenuated mutants of Newman bacterial strain by transposon insertional mutagenesis
Mus causes protective immunity.Build these mutants to block extracellular protein, the expression of surface protein and surface protein
Processing, i.e. saeR (staphylococcus aureus extracellular protein), mgrA (polygenes instrumentality A) and srtA (sorting enzyme A).But,
Although being attenuated in mice, the mutant lacking saeR or mgrA does not give the guarantor to infection of staphylococcus aureus subsequently
The immunity of protecting property.
As a result, though realize specific derivative strain virulence reduce (good attenuation), it is necessary to experimental evaluation its
Protective capability in host, so as to reach a conclusion, it can be used as live vaccine.
Summary of the invention
A first aspect of the present invention relates to a kind of preparation and comprises D-Glu Auxotrophic mutant bacterial isolates alive
The method of pharmaceutical composition (preferred vaccine), wherein said pharmaceutical composition is suitable for animal and/or the people sudden change for compositions
The prophylactic treatment (before infection) of the infection of the wild-type form of auxotrophic bacterium and/or therapeutic treatment (infect it
After Disease Clinical performance afterwards or caused by infection), and wherein said pharmaceutical composition is by the side comprised the following steps
Prepared by method:
A. D-Glu Auxotrophic mutant bacterial isolates alive is obtained;
B. bacterial isolates of being lived by described Auxotrophic mutant joins pharmaceutically acceptable carrier or diluent and appoints
Selection of land adds adjuvant;And
The most optionally pharmaceutical composition described in lyophilization.
In the preferred embodiment of a first aspect of the present invention, described preparation method comprises the following steps:
A. provide and can express glutamate racemase and possible D-amino acid transaminase and comprise peptidoglycan cell wall
Bacterial isolates;
B. it is no longer able to expressive function glutamate racemase and/or functional D-amino acid transaminase with bacterial isolates
Mode inactivates the gene of encoding glutamate racemase or multiple gene, and if it is required, inactivation codase D-aminoacid turns ammonia
The gene of enzyme or multiple gene, therefore the inactivation of wherein said gene causes described bacterial isolates to become D-Glu auxotrophy
Type;And
C. bacterial isolates of being lived by described Auxotrophic mutant joins pharmaceutically acceptable carrier or diluent and appoints
Selection of land adds adjuvant;And
The most optionally pharmaceutical composition described in lyophilization.
In another preferred embodiment or its any preferred embodiment of a first aspect of the present invention, described medicine group
Compound is vaccine, and described preparation method includes adding adjuvant.
In another preferred embodiment or its any preferred embodiment of a first aspect of the present invention, step a's) is thin
Bacteria strain is Gram-positive or gram negative bacteria.Preferably, the bacterial isolates of step a) is thin selected from consist of
Fungus kind list: Acinetobacter bauamnnii, Acinetobacter baylyi, acinetobacter calcoaceticus (Acinetobacter
Calcoaceticus), acinetobacter haemolyticus (Acinetobacter haemolyticus), Acinetobacter junii
(Acinetobacter junii), acinetobacter lwoffii (Acinetobacter lwoffii), Acinetobacter
Nosocomialis, Acinetobacter pittii, radioprotective acinetobacter calcoaceticus (Acinetobacter
Radioresistens), actinobacillus lignieresii (Actinobacillus lignieresii), actinobacillus suis
(Actinobacillus suis), Aeromonas caviae (Aeromonas caviae), Aeromonas hydrophila (Aeromonas
Hydrophila), Aeromonas veronii subsp.Sobria, actinobacillus actinomycetem comitans (Aggregatibacter
Actinomycetemcomitans), Arcobacter butzleri, Arcobacter nitrofigilis, solution starch spore
Bacillus (Bacillus amyloliquefaciens), anthrax bacillus (Bacillus anthracis), Bacillus
Bataviensis, solution fiber bacillus cereus (Bacillus cellulosilyticus), bacillus cereus (Bacillus
Cereus), Bacillus clausii (Bacillus clausii), Bacillus licheniformis (Bacillus
Licheniformis), bacillus megaterium (Bacillus megaterium), Bacillus pumilus (Bacillus
Pumilus), bacillus subtilis (Bacillus subtilis), bacillus thuringiensis (Bacillus
Thuringiensis), bacteroides fragilis (Bacteroides fragilis), Bordetella avium (Bordetella
Avium), bordetella branchiseptica (Bordetella bronchiseptica), Bordetella pertussis
(Bordetella pertusis), Bordetella petrii, Bacillus abortus (Brucella abortus), sheep kind cloth
Shandong Salmonella (Brucella melitensis), Brucella suis (Brucella suis), Burkholderia cepacia
(Burkholderia cenocepacia), glanders burkholderia (Burkholderia mallei), Burkholderia
Multivorans, Burkholderia Pseudomallei (Burkholderia pseudomallei), Burkholderia
Thailandensis, simple and clear Campylobacter spp (Campylobacter concisus), campylobacter fetus fetus subspecies
(Campylobacter fetus subsp.fetus), campylobacter fetus sexually transmitted disease (STD) subspecies (Campylobacter fetus
Subsp.venerealis), very thin Campylobacter spp (Campylobacter gracilis), Campylobacter hominis, sky
Intestinal Campylobacter spp (Campylobacter jejuni), rectum Campylobacter spp (Campylobacter rectus), Showa Campylobacter spp
(Campylobacter showae), Uppsala Campylobacter (Campylobacter upsaliensis), Freund citric acid
Bacillus (Citrobacter freundii), kirschner citric acid bacillus (Citrobacter koseri), Clostridium
Asparagiforme, bacillus botulinus (Clostridium botulinum), Bacillus butyricus (Clostridium
Butyricum), clostridium difficile (Clostridium difficile), clostridium perfringens
(Clostridium perfringens), Clostridium saccharobutylicum, Clostridium tetani
(Clostridium tetani), diphtheria corynebacterium (Corynebacterium diphtheriae), Corynebacterium pseudotuberculosis
(Corynebacterium pseudotuberculosis), clostridium perfringen (Enterobacter aerogenes), cloaca
Enterobacteria (Enterobacter cloacae), enterococcus faecalis (Enterococcus faecalis), enterococcus faecalis
(Enterococcus faecium), erysipelothrix ruhsiopathiae (Erysipelothrix rhusiopathiae), escherichia coli, bad
Dead Fusobacterium (Fusobacterium necrophorum), fusobacterium nucleatum (Fusobacterium nucleatum), adjoin
Particle chain bacterium (Granulicatella adiacens), severe foster particle chain bacterium (Granulicatella elegans), horse reproduction
Road haemophilus (Haemophilus equigenitalis), hemophilus influenza (Haemophilus influenzae), pair
Hemophilus influenza (Haemophilus parainfluenzae), haemophilus paragallinarum (Haemophilus
Paragallinarum), haemophilus parasuis (Haemophilus parasuis), pleuropneumonia haemophilus
(Haemophilus pleuropneumoniae), Haemophilus somnus (Haemophilus somnus), helicobacter pylori
(Helicobacter pylori), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella Pneumoniae
(Klebsiella pneumoniae), Oak Ridge legionella (Legionella oakridgensis), legionella pneumophilia
(Legionella pneumophila), leptospira biflexa (Leptospira biflexa), leptospira illini
(Leptospira illini), leptospria interrogans (Leptospira interrogans), monocyte hyperplasia Liszt
Bacterium (Listeria monocytogenes), capitate lysine bacillus cereus (Lysinibacillus fusiformis), spherical
Lysine bacillus cereus (Lysinibacillus sphaericus), cattle catarrhalis (Moraxella bovis), rub Gen Shi
Bacterium (Morganella morganii), mycobacterium abscessus (Mycobacterium abscessus), mycobacterium africanum
(Mycobacterium africanum), Mycobacterium avium (Mycobacterium avium), Mycobacterium bovis
(Mycobacterium bovis), Mycobacterium leprae (Mycobacterium leprae), mycobacterium tuberculosis
(Mycobacterium tuberculosis), gonococcus (Neisseria gonorrhoeae), Neisseria meningitdis
Bacterium (Neisseria meningitidis), pasteurella multocida (Pasteurella multocida), class will congratulate adjacent unit cell
Bacterium (Plesiomonas shigelloides), propionibacterium acnes (Propionibacterium acnes), Proteus
Hanseri, proteus mirabilis (Proteus mirabilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa),
Salmonella choleraesuis (Salmonella cholerasuis), Salmonella enteritidis intestinal subspecies (Salmonella enterica
Subsp.enterica), Salmonella enteritidis (Salmonella enteritidis), salmonella paratyphi
(Salmonella paratyphi), salmonella typhi (Salmonella typhi), Serratia plymuthica (Serratia
Plymuthica), shigella boydii (Shigella boydii), Shigella dysenteriae (Shigella dysenteriae), Freund
Shigella dysenteriae (Shigella flexneri), A Erlaite staphylococcus (Staphylococcus arlettae), golden yellow Portugal
Grape coccus (Staphylococcus aureus), head staphylococcus (Staphylococcus capitis), goat Fructus Vitis viniferae ball
Bacterium (Staphylococcus caprae), Staphylococcus carnosus (Staphylococcus carnosus), staphylococcus epidermidis
(Staphylococcus epidermidis), Staphylococcus equorum (Staphylococcus equorum), haemolysis Fructus Vitis viniferae ball
Bacterium (Staphylococcus haemolyticus), staphylococcus hominis (Staphylococcus hominis), road Deng's Fructus Vitis viniferae ball
Bacterium (Staphylococcus lugdunensis), Staphylococcus pasteuri (Staphylococcus pasteuri),
Staphylococcus pettenkoferi, false middle type staphylococcus (Staphylococcus
Pseudointermedius), staphylococcus saprophyticus (Staphylococcus saprophyticus), Staphylococcus
Simiae, imitation staphylococcus (Staphylococcus simulans), staphylococcus warneri (Staphylococcus
Warneri), stenotrophomonas maltophilia (Stenotrophomonas maltophilia), streptococcus agalactiae
(Streptococcus agalactiae), streptococcus dysgalactiae (Streptococcus dysgalactiae), streptococcus dysgalactiae
Like horse subspecies (Streptococcus dysgalactiae subsp.equisimilis), streptococcus equi (Streptococcus
Equi), streptococcus pneumoniae (Streptococcus pneumoniae), micrococcus scarlatinae (Streptococcus
Pyogenes), streptococcus uberis (Streptococcus uberis), Streptococcus zooepidermicus,
Taylorella asinigenitalis, horse reproductive tract Taylor Salmonella (Taylorella equigenitalis), pinta are close
Spirillum (Treponema carateum), spirochaeta cuniculi (Treponema cuniculi), Treponema
Hyodisenteriae, Tyreponema pallidum (Treponema pallidum), Treponema suis, Veillonella atypica
(Veillonella atypica), different Veillonella (Veillonella dispar), veillonella parvula (Veillonella
Parvula), rat Veillonella (Veillonella ratti), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious
(Vibrio parahaemolyticus), Vibrio vulnificans, Yersinia enterocolitica (Yersinia
Enterocolitica), Yersinia pestis (Yersinia pestis) and yersinia pseudotuberculosis (Yersinia
pseudotuberculosis)。
It is highly preferred that the list of the bacterial isolates choosing freely following species composition of described step a): Acinetobacter bauamnnii, copper
Green pseudomonas and staphylococcus aureus.It is highly preferred that described bacterial isolates is the bacterial isolates of Acinetobacter bauamnnii, its life
Entitled Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor Novoa Santos in cloth
It is deposited in Spain's Type Tissue Collection with strain number 8588 on April 14th, 2014 under Da Peisi treaty.More excellent
Selection of land, described bacterial isolates is the bacterial isolates of Pseudomonas aeruginosa (P.aeruginosa), its named Pseudomonas aeruginosa
DeltaPA4662, and by Fundaci ó n Profesor Novoa Santos under budapest treaty with strain number
8589 are deposited in Spain's Type Tissue Collection on April 14th, 2014.It is highly preferred that described bacterial isolates is golden yellow
The staphylococcic bacterial isolates of color, its named 132deltamurI/deltadat, and by Fundaci ó n Profesor
Novoa Santos is deposited in Spain typical case with strain number 8587 on June 11st, 2014 under budapest treaty and cultivates
Thing preservation center.
A second aspect of the present invention relates to a kind of pharmaceutical composition, and preferably a kind of vaccine, it comprises D-Glu nutrition and lacks
Swaged mutant is lived bacterial isolates and pharmaceutically acceptable carrier or diluent and the adjuvant being optionally present, wherein said medicine
Compositions is suitable for animal and/or people infection pre-for the wild-type form of the sudden change auxotrophic bacterium of described compositions
Anti-property (before infection) and/or therapeutic treatment (after the clinical manifestation of the disease after infection or caused by infection).
In a preferred embodiment of a second aspect of the present invention, described pharmaceutical composition is vaccine, and described epidemic disease
Seedling optionally comprises adjuvant.
In the another embodiment or its any preferred embodiment of a second aspect of the present invention, described pharmaceutically acceptable
Carrier or diluent selected from the list that consists of: water, culture fluid, the solution of physiological salt concentration and/or stabilizer are such as
SPGA, carbohydrate (such as sorbitol, mannitol, starch, sucrose, glucose, glucosan), albumen such as albumin or cheese egg
In vain, protein-contg material such as Ox blood serum or skimmed milk and buffer (such as phosphate buffer) are wrapped.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described adjuvant selects
List from consisting of: Freund completely and Freund's incomplete adjuvant, vitamin E, non-ionic block polymer, muramyldipeptide,
ISCOM (immunostimulating complex), saponin, mineral oil, vegetable oil, Carbopol, escherichia coli (E.coli) heat-labile toxin
(LT) or cholera toxin (CT), aluminium hydroxide, aluminum phosphate or aluminium oxide, oil-Emulsion (such asOrSaponin and Viamin E solubilisate.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described medicine group
It is 10 that compound comprises dosage3-1010The D-Glu Auxotrophic mutant of individual antibacterial bacterial isolates alive.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described medicine group
Compound is freeze-dried.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described Bacteria
Select good strains in the field for seed from the bacterial species list consisted of: Acinetobacter bauamnnii, Acinetobacter baylyi, calcium acetate not lever
Bacterium, acinetobacter haemolyticus, Acinetobacter junii, acinetobacter lwoffii, Acinetobacter nosocomialis,
Acinetobacter pittii, radioprotective acinetobacter calcoaceticus, actinobacillus lignieresii, actinobacillus suis, Aeromonas caviae, addicted to water
Aeromonas, Aeromonas veronii subsp.sobria, actinobacillus actinomycetem comitans, Arcobacter butzleri,
Arcobacter nitrofigilis, bacillus amyloliquefaciens, anthrax bacillus, Bacillus bataviensis, solution fibrous bud
Spore bacillus, bacillus cereus, Bacillus clausii, Bacillus licheniformis, bacillus megaterium, Bacillus pumilus, withered
Grass bacillus cereus, bacillus thuringiensis, bacteroides fragilis, Bordetella avium, bordetella branchiseptica, pertussis are won
Moral spy Salmonella, Bordetella petrii, Bacillus abortus, brucella melitensis, Brucella suis, primary gram of Hall of Bulbus Allii Cepae
Moral bacterium, glanders burkholderia, Burkholderia multivorans, Burkholderia Pseudomallei,
Burkholderia thailandensis, simple and clear Campylobacter spp, campylobacter fetus fetus subspecies, campylobacter fetus sexually transmitted disease (STD) subspecies,
Very thin Campylobacter spp, Campylobacter hominis, campylobacter jejuni, rectum Campylobacter spp, Showa Campylobacter spp, Uppsala are curved
Curved bar bacterium, citrobacter freundii, kirschner citric acid bacillus, Clostridium asparagiforme, bacillus botulinus, butyric acid
Clostruidium, clostridium difficile, clostridium perfringens, Clostridium
Saccharobutylicum, Clostridium tetani, diphtheria corynebacterium, Corynebacterium pseudotuberculosis, clostridium perfringen,
Enterobacter cloacae, enterococcus faecalis, enterococcus faecalis, erysipelothrix ruhsiopathiae, escherichia coli, actinomyces pseudonecrophorus, fusobacterium nucleatum, adjoin
Particle chain bacterium, severe foster particle chain bacterium, horse reproductive tract haemophilus, hemophilus influenza, haemophilus parainfluenzae, the secondary bloodthirsty bar of chicken
Bacterium, haemophilus parasuis, pleuropneumonia haemophilus, Haemophilus somnus, helicobacter pylori, Klebsiella oxytoca, pneumonia
Klebsiella, Oak Ridge legionella, legionella pneumophilia, leptospira biflexa, leptospira illini, kidney hook end spiral
Body, Listeria monoeytogenes, capitate lysine bacillus cereus, spherical lysine bacillus cereus, cattle catarrhalis, rub root
Salmonella, mycobacterium abscessus, mycobacterium africanum, Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae, tuberculosis branch bar
Bacterium, gonococcus, Neisseria meningitidis, pasteurella multocida, Plesiomonas shigelloides, propionibacterium acnes,
Proteus hanseri, proteus mirabilis, Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella enteritidis intestinal subspecies,
Salmonella enteritidis, salmonella paratyphi, salmonella typhi, Serratia plymuthica, shigella boydii, Shigella dysenteriae, not
Family name's shigella dysenteriae, A Erlaite staphylococcus, staphylococcus aureus, head staphylococcus, Staphylococcus caprae, meat Fructus Vitis viniferae ball
Bacterium, staphylococcus epidermidis, Staphylococcus equorum, staphylococcus haemolyticus, staphylococcus hominis, road Deng's staphylococcus, Pasteur's Fructus Vitis viniferae ball
Bacterium, Staphylococcus pettenkoferi, false middle type staphylococcus, staphylococcus saprophyticus, Staphylococcus
Simiae, imitate staphylococcus, staphylococcus warneri, stenotrophomonas maltophilia, streptococcus agalactiae, streptococcus dysgalactiae, stop breast
Streptococcus is like horse subspecies, streptococcus equi, streptococcus pneumoniae, micrococcus scarlatinae, streptococcus uberis, Streptococcus
Zooepidermicus, Taylorella asinigenitalis, horse reproductive tract Taylor Salmonella, treponema carateum, rabbit
Treponema, Treponema hyodisenteriae, Tyreponema pallidum, Treponema suis, Veillonella atypica,
Different Veillonella, veillonella parvula, rat Veillonella, vibrio cholera, vibrio parahaemolytious, Vibrio vulnificans,
Yersinia enterocolitica, Yersinia pestis and yersinia pseudotuberculosis.
It is highly preferred that the list of the bacterial isolates choosing freely following species composition of described step a): Acinetobacter bauamnnii, copper
Green pseudomonas and staphylococcus aureus.It is highly preferred that described bacterial isolates is the bacterial isolates of Acinetobacter bauamnnii, its life
Entitled Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor Novoa Santos in cloth
It is deposited in Spain's Type Tissue Collection with strain number 8588 on April 14th, 2014 under Da Peisi treaty.More excellent
Selection of land, described bacterial isolates is the bacterial isolates of Pseudomonas aeruginosa, its named Pseudomonas aeruginosa DeltaPA4662, and
And by Fundaci ó n Profesor Novoa Santos under budapest treaty with strain number 8589 in April, 2014
Within 14th, it is deposited in Spain's Type Tissue Collection.It is highly preferred that described bacterial isolates is the thin of staphylococcus aureus
Bacteria strain, its named 132deltamurI/deltadat, and by Fundaci ó n Profesor Novoa Santos in
It is deposited in Spain's Type Tissue Collection with strain number 8587 on June 11st, 2014 under budapest treaty.
A third aspect of the present invention relates to D-Glu Auxotrophic mutant bacterial isolates alive, wherein said Bacteria
Strain is the bacterial isolates of Pseudomonas aeruginosa, its named Pseudomonas aeruginosa DeltaPA4662, and by Fundaci ó n
Profesor Novoa Santos is deposited in western class with strain number 8589 on April 14th, 2014 under budapest treaty
Tooth Type Tissue Collection.
A third aspect of the present invention further relates to D-Glu Auxotrophic mutant bacterial isolates alive, wherein said antibacterial
Bacterial strain is the bacterial isolates of staphylococcus aureus, its named 132deltamurI/deltadat, and by Fundaci ó n
Profesor Novoa Santos is deposited in western class with strain number 8587 on June 11st, 2014 under budapest treaty
Tooth Type Tissue Collection.
A fourth aspect of the present invention relates to the bacterial isolates as defined in a third aspect of the present invention, as medicine, especially
It is used as vaccine.
A fifth aspect of the present invention relates to the pharmaceutical composition of a second aspect of the present invention or the 3rd of the present invention or
The D-Glu Auxotrophic mutant of four aspects bacterial isolates alive, the dashing forward for described compositions for animal and/or people
The prophylactic treatment (before infection) and/or the therapeutic treatment that become the infection of the wild-type form of auxotrophic bacterium (infect
After the clinical manifestation of the disease afterwards or caused by infection) method.
A sixth aspect of the present invention relates to antibody or it is selected from Fab, F (ab') 2, the sheet of Fv, scFv, di-scFv and sdAB
Section, it is capable of identify that D-Glu Auxotrophic mutant bacterial isolates alive, and wherein said antibody or its fragment are suitable for animal
And/or people is for the prophylactic treatment (infection of the infection of the wild-type form of the sudden change auxotrophic bacterium of described compositions
Before) and/or therapeutic treatment (after infection or infect caused by disease clinical manifestation after).
A seventh aspect of the present invention relates to antibody or it is selected from Fab, F (ab') 2, the sheet of Fv, scFv, di-scFv and sdAB
Section, it obtains after bacterial isolates immunising mammals of living with D-Glu Auxotrophic mutant and maybe can obtain, wherein
Described antibody or its fragment are suitable for animal and/or the people wild-type form for the sudden change auxotrophic bacterium of described compositions
The prophylactic treatment (before infection) of infection and/or the therapeutic treatment (clinic of the disease after infection or caused by infecting
After performance).
A eighth aspect of the present invention relates to a kind of pharmaceutical composition, preferably a kind of vaccine, its comprise the present invention the 6th or
The antibody of either side or its fragment and pharmaceutically acceptable carrier or diluent and the adjuvant being optionally present in 7th aspect,
Wherein said pharmaceutical composition is suitable for animal and/or the people wild type shape for the sudden change auxotrophic bacterium of described compositions
Preventative (before the infection) of the infection of formula and/or therapeutic treatment (the clinical table of the disease after infection or caused by infection
After now).In a preferred embodiment of a eighth aspect of the present invention, described pharmaceutical composition is vaccine, wherein said epidemic disease
Seedling optionally comprises adjuvant.
A ninth aspect of the present invention relates to antibody or its fragment of the 6th or the 7th aspect of the present invention, is used for treating, special
It is not for passive immunity.
A tenth aspect of the present invention relates to the pharmaceutical composition of a eighth aspect of the present invention or the 6th of the present invention or
The antibody of seven aspects or its fragment, for animal and/or people for sudden change auxotrophic bacterium wild of described compositions
The prophylactic treatment (before infection) of the infection of type form and/or the therapeutic treatment (disease after infection or caused by infection
Clinical manifestation after) method.
A eleventh aspect of the present invention relates to the second of the present invention or the pharmaceutical composition of eighth aspect or the present invention
The D-Glu Auxotrophic mutant of the third aspect live bacterial isolates or the present invention the 6th or the 7th aspect in arbitrary
The antibody of aspect or its fragment, for animal and/or people for the wild type of the sudden change auxotrophic bacterium of described compositions
The prophylactic treatment (before infection) of the infection of form and/or therapeutic treatment be (disease after infection or caused by infecting
After clinical manifestation) method, and wherein said compositions, bacterial isolates or antibody or its fragment intranasal, Intradermal, subcutaneous,
It is administered orally, is administered by aerosol, intramuscular, wing lattice (wing web) and eye drop.
Additionally, the author of the present invention is surprisingly found that the reagent of antibody or its fragment comprising the present invention by use
Box or device, described test kit allows the most qualitative and/or that quantitative analysis is individual biological sample, particularly suspects and suffer from carefully
Bacterial species in the individual blood plasma of bacterium original disease.
Therefore, a twelveth aspect of the present invention relates to a kind of test kit or device, and it comprises the 7th or the 8th of the present invention the
The antibody of either side or its fragment in aspect.
The preferred embodiment of a twelveth aspect of the present invention relates to a kind of for detecting bacterial origin by immunoassay
The test kit infected or device, it comprises:
The first antibody being referred to as " capture antibody " of either side definition in terms of the 6th or the 7th of (i) such as the present invention,
Wherein said first antibody is preferably attached to solid support;
(ii) being referred to as second traget antibody of " detection antibody ", it identifies the district in addition to the region of first antibody identification
Territory, wherein said second antibody comprises labelling, and described labelling can be fluorescence, shiner or enzyme;
(iii) show the reagent of the affinity to second antibody, described reagent be coupled to combine to the first member;With
And
(iv) combine to the second member, it is coupled to fluorescence, shiner or enzyme, and wherein said combination is to being selected from: half
Antigen and antibody;Antigen and antibody;Biotin and avidin;Biotin and Succ-PEG-DSPE;Biotin class
Like thing and avidin;Biotin analog and Succ-PEG-DSPE;Sugar and agglutinin;Enzyme and cofactor;Nucleic acid
Or nucleic acid analog and complementary nucleic acid or nucleic acid analog.
A thirteenth aspect of the present invention relates to the test kit of a twelveth aspect of the present invention or the purposes of device, for qualitative
And/or quantitatively determine the biological sample from mammal, particularly suspect in the blood plasma of the mammal suffering from bacterial disease
Bacterial species or bacterial isolates.
A fourteenth aspect of the present invention relates to a kind of method cultivating the auxotrophic bacterial isolates of D-Glu, described
Method includes the D-Glu concentration utilizing 0.00001-120mM.Preferably, the concentration range of D-Glu is 0.01-50mM.
It is highly preferred that the concentration range of D-Glu is 10-20mM.
Accompanying drawing explanation
Fig. 1 schematically shows the general structure (not shown teichoic acid and albumen) of the bacteria wall of gram-positive bacterium.
PG: Peptidoglycan (murein).M: cytoplasma membrane.
Fig. 2 schematically shows structure and composition (not shown lipopolysaccharide and the egg of the bacteria wall of gram negative bacteria
In vain).PG: Peptidoglycan (murein).OM: adventitia.IM: inner membrance.
Fig. 3 schematically shows and forms and be incorporated to metabolic process that bacteria cell wall Peptidoglycan terminates with D-Glu
Sequentially.Dat, murI and murD gene shown in figure is separately encoded Dat, MurI and MurD albumen.
Fig. 4 illustrate utilize Clustal Omega program Acinetobacter bauamnnii ATCC 17978 (A3MIP5_ACIBT and
A3MA43_ACIBT) 2 kinds of MurI glutamate racemases, escherichia coli (MURI_ECOLI) and Pseudomonas aeruginosa (MURI_
The comparison of aminoacid sequence PSEAE).Residue dark gray background identical in all glutamate racemases illustrates.
Fig. 5 illustrates in Acinetobacter bauamnnii ATCC 17978 mutant Δ 0380, Δ 3398 and Δ 0380/ Δ 3398 and lacks
PCR confirm.A: oligonucleotide EXTFW0380 and EXTRV0380 is used for from the bacterial strain carrying wild type gene seat A1S_0380
Produce and there is the fragment of 1116bp or produce 345bp fragment from the bacterial strain carrying mutant gene seat Δ 0380.B: oligonucleotide
EXTFW3398 and EXTRV3398 is used for producing the fragment with 1056bp from the bacterial strain carrying wild type gene seat A1S_3398
Or produce 516bp fragment from the bacterial strain carrying mutant gene seat Δ 3398.DNA fragmentation in Mei Tiao road has a following coupling:
MW, molecular weight modes;0380, it is produced from the amplicon of the bacterial strain carrying wild type gene seat A1S_0380;3398, it is produced from and takes
The amplicon of the bacterial strain with wild type gene seat A1S_3398;Δ 0380/ Δ 3398, is produced from two locus A1S_0380
With the amplicon of the bacterial strain of defect in A1S_3398;Δ 3398 and Δ 0380, originate from locus A1S_3398 and
The amplicon of the bacterial strain of defect in A1S_0380.
Fig. 6 illustrates during structure Acinetobacter bauamnnii ATCC 17978 double-mutant Δ 0380/ Δ 3398 caused by cointegrate
The screening of bacterium colony.From the LB agar with 15% sucrose and 10mM D-Glu select single bacterium colony and be seeded in or without
Same position in the LB agar of 10mM D-Glu.The bacterium colony with Δ 0380/ Δ 3398 genotype only has D-paddy ammonia
The flat board of acid grows;The bacterium colony with Δ 0380 genotype all grows with or without D-Glu.
Fig. 7 illustrates Acinetobacter bauamnnii wild-type strain ATCC 17978 and the life of double mutant strain Δ 0380/ Δ 3398
Long and viability measures.Bacterial strain Δ 0380/ Δ 3398 shows normal raw in supplementing the culture medium of D-Glu of 10mM
Long, but do not have the external source supply of this compound then can not grow.By contrast, wild-type strain is adding or is being not added with D-
All according to normal growth in the LB culture medium of glutamic acid.In each figure, filled squares (■) represents the training having D-Glu
Supporting the wild-type strain in base, open squares () represents the wild-type strain not having D-Glu, solid circles (●) generation
Table has a double-mutant in the culture medium of D-Glu, and that hollow circle (zero) represents in the culture medium not having D-Glu is double
Mutant.A: double-mutant and wild-type strain cultivate optical density.B: double-mutant and wild-type strain cultivate viability
(Log10CFU/mL)。
Fig. 8 is shown in the presence of the D-Glu of variable concentrations, at Acinetobacter bauamnnii ATCC17978 Δ 0380/ Δ
Relative to its wild type homologue in cell division and the difference of Morphology level in 3398 double-mutants.Use scanning electron microscopy
Mirror shooting microphotograph.A: two kinds of bacterial strains are all cultivated under the D-Glu increasing concentration, and aobvious with the shooting of same ratio chi
The micro-photo scale of 10 μm (bar represent);B: two kinds of bacterial strains are all cultivated in the presence of 0.1mM D-Glu, and to pass
The scale subtracting order progressively shoots microphotograph.
Fig. 9 is shown in the presence of keeping D-Glu not, Acinetobacter bauamnnii ATCC 17978 mutant strain Δ 0380/ Δ
The cell wall of 3398 and the different atypical morphology of cracking, Step denaturation.Clap with transmission electron microscope chi in varing proportions
Take the photograph microphotograph.Black arrow instruction has normal morphology or the whole bacterial cells of atypical cell division;Dotted arrow
Instruction does not has the content of the smudge cells of bacteria cell wall, cell lysis or confusion, scattered hereditary material, film and/or fat
The gathering of plastid.
Figure 10 illustrates the Acinetobacter bauamnnii wild-type strain ATCC 17978 (A) by various dose and Δ 0380/ Δ
After 3398 mutant strains (B) intraperitoneal injection, the percentage survival (n=6) of BALB/c mouse is to determine 100% susceptible mice
Fatal dose (the LD) (LD that meeting is dead100)(A)LD100=2.5X;(B)LD100=6X.Mouse survival is monitored in 7 days.
Figure 11 illustrates with the Acinetobacter bauamnnii wild-type strain ATCC 17978 of 2X dosage, bacterial strain Δ 0380, bacterial strain Δ
3398 and bacterial strain Δ 0380/ Δ 3398 infect after 12 hours, the bacterial load (n=8-9 mice) in BALB/c mouse liver.
P-value is checked according to Mann-Whitney U.Each point represents the independent bacterial load of Mouse Liver, and each horizontal line represents
Often organize respective meansigma methods.
Figure 12 illustrates mice at the 0th and 14 day with bacterial strain Δ 0380/ Δ 3398 preimmunization or the most immune (saline pair
According to) after, with the 21st day be administered 4X dosage Acinetobacter bauamnnii wild-type strain ATCC 17978 infect after 12 hours
Bacterial load (n=10) in the liver of BALB/c mouse, spleen and lung.P-value is checked according to Mann-Whitney U.Each some generation
The independent bacterial load of table mouse organs.The meansigma methods often organized is represented by horizontal line.
Figure 13 immunity inoculation is shown after the 7th and 21 day right in BALB/c mouse (n=12) with in non-immunity inoculation
Log according to the IgG antibody produced for Acinetobacter bauamnnii strains A TCC 17978 in mice (saline control)101/ terminal drips
Degree.Antibody titer is determined by indirect ELISA.* P < 0.0001 compared with the mice group of non-immunity inoculation;After immunity inoculation
#P < 0.0240 is compared in the generation of the 7th day IgG;P-value is checked according to Mann-Whitney U.Square frame represents first and the three or four point
Figure place;Horizontal line represents median;Scope must be represented by line (whisker).
Figure 14 illustrates after immunity inoculation the 21st day by Acinetobacter bauamnnii strains A TCC 17978 of various dose
(0.01X;0.05X;0.1X;0.5X and 1X) BALB/c mouse (n=6) in and in the control mice (0X) of non-immunity inoculation
Log for the IgG antibody that Acinetobacter bauamnnii strains A TCC 17978 produces101/ end point titres.True by indirect ELISA
Determine antibody titer.* P < 0.05 compared with the mice group of non-immunity inoculation;P-value is checked according to Mann-Whitney U.Square frame generation
Table first and the 3rd quartile;Horizontal line represents median;Scope must be represented by line.
Figure 15 immunity inoculation is shown after the 21st day BALB/c mouse and the control mice (saline control) of non-immunity inoculation
The IgG antibody produced is for Acinetobacter bauamnnii bacterial strain: ATCC 17978, the cross reactivity of ATCC19606 and AbH12O-A2
(titre).
Figure 16 is BALB/c after the Acinetobacter bauamnnii ATCC 17978 wild-type strain intra-abdominal infection with 4X dosage
The percentage survival (n=12) of mice.By the mice of immunity inoculation at the 0th and 14 day with Acinetobacter bauamnnii Δ 0380/ Δ
3398 bacterial strain immunity, and infected with wild-type strain at the 21st day.It is administered salt to the mice of non-immunity inoculation at the 0th and 14 day
Water, and infecting with wild-type strain on the same day.* the survival P < 0.0001 of immunity inoculation group compared with matched group.P-value, root
(log-rank inspection) is checked according to Mantel-Cox.
Figure 17 is with the depositing of BALB/c mouse after the Acinetobacter bauamnnii AbH12O-A2 bacterial strain intra-abdominal infection of 4X dosage
Percentage ratio (n=9) alive.The mice of immunity inoculation was exempted from Acinetobacter bauamnnii Δ 0380/ Δ 3398 bacterial strain at the 0th and 14 day
Epidemic disease, and infected by clinical strains at the 21st day.It is administered saline to the mice of non-immunity inoculation at the 0th and 14 day, and same
Within one day, infect by clinical strains.* the survival P < 0.0001 of immunity inoculation group compared with matched group.P-value, according to Mantel-Cox
Inspection (log-rank inspection).
After Figure 18 is the encapsulated Acinetobacter bauamnnii Ab307-0294 bacterial strain intra-abdominal infection with 0.75X dosage
The percentage survival (n=6-8) of BALB/c mouse.The mice of immunity inoculation was used Acinetobacter bauamnnii Δ at the 0th and 14 day
0380/ Δ 3398 bacterial strain immunity, and infected with supper toxic strain at the 21st day.The the 0th and 14 day little to non-immunity inoculation
Mus is administered saline, and is using Ab307-0294 strain infection on the same day.* the survival P=of immunity inoculation group compared with matched group
0.0022.P-value, checks (log-rank inspection) according to Mantel-Cox.
Figure 19 is the Bao Man not lever recovered during growth under 37 DEG C with stirring (180rpm) in distilled water in 40 days
Bacterium ATCC 17978 wild type and the Log of Δ 0380/ Δ 3398 bacterial strain10CFU/mL.By counting plating at LB (wild type
Bacterial strain) and the bacterium colony that supplements on the LB (mutant strain) of 10mM D-Glu to determine CFU.All cultivations are the most triplicate
Carry out.
Figure 20 be in 8 days in 37 DEG C with stirring (180rpm) under by this strain culturing to supplementing 10mM D-paddy ammonia
From LB (zero) with supplement Acinetobacter bauamnnii Δ 0380/ Δ 3398 that the LB (●) of 10mM D-Glu recovers during the LB of acid
The quantity (CFU/mL) of bacterium colony.
Figure 21 illustrates over time from 100 μ L Acinetobacter bauamnnii ATCC 17978 wild type and Δ 0380/ Δs 3398
Quantity (the Log of the Acinetobacter bauamnnii bacterium colony that the mouse blood that bacterial strain (1X dosage) is inoculated recovers10CFU/mL)。
Figure 22 illustrates the screening of the cointegrate determined during the Δ PA4662 mutant building Pseudomonas aeruginosa.
Figure 23 illustrates that the PCR lacked in the Δ PA4662 mutant of Pseudomonas aeruginosa confirms.Primer EXTFWPA4662 and
EXTRVPA4662 is used for from carrying wild type PA4662 allelic bacterial strain generation 1741bp fragment or from carrying Δ PA4662
Allelic bacterial strain produces 943bp fragment.The sample of swimming lane labelling and analysis is as follows: MW:DNA scalariform band;Δ PA4662:
Amplicon from the bacterial strain carrying Δ PA4662 mutation allele;And PA4662: from carrying wild type PA4662 etc.
The amplicon of the bacterial strain of position gene.
Figure 24 illustrates Pseudomonas aeruginosa wild-type strain PAO1 (A) by various dose and Δ PA4662 mutant strain
(B) after intraperitoneal injection, the percentage survival (n=4) of BALB/c mouse is lethal with determine that 100% susceptible mice can be dead
Dosage (LD) (LD100)(A)LD100=0.4X;(B)LD100>40X.Mouse survival is monitored in 7 days.
Figure 25 be shown in holding D-Glu not in the presence of, the cell wall of Pseudomonas aeruginosa mutant strain Δ PA4662 and
The atypical morphology of cracking and Step denaturation.A-E:MH culture medium;F-I:LB culture medium;J-T:LB+MgCl2(30mg/L)+
CaCl2(75mg/L).Microphotograph is shot with transmission electron microscope chi in varing proportions.Black arrow instruction has normal shape
State or the whole bacterial cells of atypical cell division;Dotted arrow instruction does not has the smudge cells of bacteria cell wall, cracking
Cell or the content of confusion, scattered hereditary material, film and/or the gathering of liposome.
Figure 26 is BALB/c mouse after the Pseudomonas aeruginosa PAO1 wild-type strain intra-abdominal infection with 0.4X dosage
Percentage survival (n=8).By the mice of immunity inoculation at the 0th and 14 day with Pseudomonas aeruginosa Δ PA4662 bacterial strain (0.4X
Dosage A;0.04X dosage B) immunity, and infected with wild-type strain at the 25th day.At the 0th and 14 day to non-immunity inoculation
Mice be administered saline, and infecting with wild-type strain on the same day.* compared with matched group immunity inoculation group survival P <
0.0001.P-value, checks (log-rank inspection) according to Mantel-Cox.
Figure 27 be intraperitoneal injection 0.4X dosage Pseudomonas aeruginosa PAO1 wild-type strain after BALB/c mouse
Percentage survival (n=8).In (A), before infecting, mice vaccine serum (producing with Δ PA4662 vaccine) is passively exempted from
Epidemic disease or be administered inmature serum, in (B), at the rear vaccine serum being administered two dosage to mice of the development of acute septicopyemia symptom
(producing with Δ PA4662 bacterial strain) or inmature serum.* compared with the mice accepting inmature serum, with vaccine serum passive immunity
The survival P < 0.05 of mice.P-value, checks (log-rank inspection) according to Mantel-Cox.
Figure 28 is the P. aeruginosa recovered during growth under 37 DEG C with stirring (180rpm) in distilled water in 157 days
Bacterium PAO1 wild type and the Log of Δ PA4662 bacterial strain10CFU/mL.And had at LB (wild-type strain) by counting plating
Bacterium colony on the LB (mutant strain) of 10mM D-Glu determines CFU.All cultivations are carried out the most in triplicate.
Figure 29 be in 5 days in 37 DEG C with stirring (180rpm) under by this strain culturing to having 10mM D-Glu
LB time from LB (zero) and the number that supplements the Pseudomonas aeruginosa Δ PA4662 bacterium colony that the LB (●) of 10mM D-Glu recovers
Amount (CFU/mL).
Figure 30 be shown in structure staphylococcus aureus double-mutant Δ murI/ Δ dat during second time crossing event it
The screening of the bacterium colony caused by rear single mutant Δ murI.Select individually from the TSB agar plate supplementing X-Gal (150 μ g/mL)
Erythromycin-sensitive bacterium colony and be seeded in or without the same position in the TSB agar plate of 10mM D-Glu.There is Δ
The bacterium colony of murI/ Δ dat genotype only grows in the flat board have D-Glu, and the bacterium colony with Δ murI genotype (lacks
Lack murI gene but there is the dat gene of complete copy) with or without the equal normal growth of D-Glu.
Figure 31 is that in staphylococcus aureus 132 mutant Δ murI, Δ dat and Δ murI/ Δ dat, the PCR of disappearance is true
Recognize.The primer used is: A:murIF/murIR;B:murIExtF/murIExtR;C:murIseqF/murIseqR;D:datF/
datR;E:datExtF/datExtR;And F:datseqF/datseqR.Swimming lane: MM, molecular marker GeneRuler 1kb;Δ
MurI, it is thus achieved that certainly carry the fragment of the bacterial strain of mutant gene seat Δ murI;Δ dat is derived from and carries mutant gene seat Δ dat's
The fragment of bacterial strain;Δ murI/ Δ dat is derived from the fragment of the bacterial strain of two locus Δ murI and Δ dat defect;Wild type,
It is derived from the fragment of staphylococcus aureus 132 wild-type strain carrying two locus murI and dat.
Figure 32 illustrates staphylococcus aureus 132 wild type and the growth of double mutant strain Δ murI/ Δ dat and viability
Measure.Bacterial strain Δ murI/ Δ dat shows normal growth in supplementing the TSB of D-Glu of 20mM, but the most this
The external source supply of compound then can not grow.By contrast, wild-type strain is adding or is being not added with in the TSB of D-Glu raw
Long.Each symbology a kind of bacterial strain as For the illustrated example.A: cultivate turbidity;B: cultivate viability.
Figure 33 is shown in the presence of the D-Glu of variable concentrations, at staphylococcus aureus 132 double-mutant Δ
Relative to its S. aureus wild type bacterial strain in the change of Morphology level in murI/ Δ dat.Use scanning electron microscopy
Mirror shooting image.A: two kinds of bacterial strains are all cultivated under the D-Glu increasing concentration, and shoot micro-photograph with same ratio chi
The sheet scale of 10 μm (horizontal bar represent);B: two kinds of bacterial strains are all cultivated in the culture medium supplementing 0.1mM D-Glu,
And progressively shoot image with the scale of order of successively decreasing.
Figure 34 is shown in the presence of keeping D-Glu not, 132 pairs of mutant strain Δ murI/ Δs of staphylococcus aureus
The cell wall of dat and the different atypical morphology of cracking, Step denaturation.Such as the chi transmission in varing proportions described in horizontal bar
Ultramicroscope shooting image.Black arrow instruction has normal morphology or the whole bacterial cells of atypical cell division;
Dotted arrow instruction does not has the content of the smudge cells of bacteria cell wall, cell lysis or confusion, scattered hereditary material, film
And/or the gathering of liposome.
Figure 35 illustrates the staphylococcus aureus 132 wild type (A) by various dose and Δ murI/ Δ dat mutant (B)
After intraperitoneal injection, the percentage survival (n=3-4) of BALB/c mouse is to determine the lethal agent that 100% susceptible mice can be dead
Amount (LD) (LD100)(A)LD100=3X;(B)LD100>30X.Mouse survival is monitored in 14 days.
Figure 36 illustrates mice at the 0th and 14 day with the bacterial strain Δ murI/ Δ dat preimmunization or the most immune of 10X dosage
After (saline control), 20 hours BALB/c mouse after infecting with staphylococcus aureus 132 wild-type strain of 5X dosage
Spleen (A) and blood cultures (B) in bacterial load (n=4-6).Each point represents the independent bacterial load of mice spleen.Often
The meansigma methods of group is represented by horizontal line.P=0.0095 compared with the mice group of non-immunity inoculation.P-value is according to Mann-
Whitney U checks.Blood cultures from every mice incubation at 37 DEG C is not shaken for 18 hours: (+), positive;
(-), negative.
Figure 37 illustrates 22 hours BALB/c mouse after infecting with staphylococcus aureus 132 wild-type strain of 5X dosage
Spleen (A) and blood (B) in bacterial load (n=8-9).By mice at the 0th and 14 day with the bacterial strain Δ murI/ Δ of 8X dosage
Dat immune or the most immune (saline control).Each point represents the independent bacterial load of mice spleen or blood.The meansigma methods often organized is led to
Cross horizontal line to represent.P=0.0006 and P=0.0002 compared with the mice group of non-immunity inoculation.P-value is according to Mann-
Whitney U checks.
Figure 38 is BALB/c mouse after the staphylococcus aureus 132 wild-type strain intra-abdominal infection with 5X dosage
Percentage survival (n=10-13).The mice of immunity inoculation was exempted from the Δ murI/ Δ dat bacterial strain of 10X dosage at the 0th and 14 day
Epidemic disease, is being administered saline to non-immune mice simultaneously on the same day.* P=0.031 compared with the mice group of non-immunity inoculation.P-value
Check according to Mann-Whitney U.Monitoring mouse survival is until 96 hours.
Figure 39 illustrates the BALB/c mouse (n=8-10) with the Δ murI/ Δ dat bacterial strain preimmunization of 10X dosage and not
For the Log of the IgG antibody waiting gene staphylococcus aureus 132 Δ spa bacterial strain to produce in the mice (saline control) of immunity10
1/ end point titres.Antibody titer is determined by indirect ELISA.* P < 0.0001 compared with the mice group of non-immunity inoculation.P-value root
Check according to Mann-Whitney U.Square frame represents first and the 3rd quartile;Horizontal line represents median;Model must be represented by line
Enclose.
Figure 40 illustrates the BALB/c mouse (n=6-9) with the Δ murI/ Δ dat bacterial strain preimmunization of 10X dosage and rather
The IgG antibody produced in the mice (saline control) of epidemic disease is for the staphylococcus aureus strains of separate sources: (A)
USA300LAC (people);(B) RF122 (cattle);(C) ED133 (sheep);(D) cross reactivity (Log of ED98 (poultry)101/ eventually
Point titre).Antibody titer is determined by indirect ELISA.Compared with the mice group of non-immunity inoculation#P < 0.0001 and * P <
0.0006.P-value is checked according to Mann-Whitney U.Square frame represents first and the 3rd quartile;Position in horizontal line representative
Number;Scope must be represented by line.
Figure 41 is shown in 5 days the golden yellow Fructus Vitis viniferae recovered during growth under room temperature with stirring (180rpm) in distilled water
Coccus wild type and the Log of Δ murI/ Δ dat bacterial strain10CFU/mL.By counting plating at TSB agar (wild type) and
Supplement the bacterium colony on the TSB (double mutant strain) of 10mM D-Glu to determine CFU.All cultivations are carried out the most in triplicate.
Figure 42 be shown in 11 days in 37 DEG C with stirring (180rpm) under by this strain culturing to supplementing 20mM D-paddy
From TSB (zero) with supplement the staphylococcus aureus Δ murI/ that the TSB (●) of 10mM D-Glu recovers during the TSB of propylhomoserin
The quantity (CFU/mL) of Δ dat bacterium colony.
Figure 43 illustrates over time, with staphylococcus aureus 132 wild-type strain and the 10X agent of sub-lethal 0.7X dosage
Amount the kidney (CFU/g) (A) of mice (n=3/ often group) of Δ murI/ Δ dat bacterial strain intraperitoneal inoculation, spleen (CFU/g) (B) and
The S. aureus colonies quantity recovered in blood sample (CFU/ml) (C).Infect after 1,2 and 6 days little by one
Mus (often group/every bacterial strain) euthanasia.For each bacterial strain, the CFU/ mice inoculated when 0 shown in Y-axis.At TSB agar
(wild-type strain) or TSB agar are plus recovering bacterium colony in 10mM D-Glu (Δ murI/ Δ dat bacterial strain).
Figure 44 illustrates with the pseudomonas aeruginosa strains PAO1 (0.1X of various dose;0.4X;1X;4X;10X and 40X) exempt from
Epidemic disease inoculation after the 40th day in BALB/c mouse and in the control mice (0X) of non-immunity inoculation for Pseudomonas aeruginosa
The end point titres Log of the IgG antibody that bacterial strain PAO1 produces10.Antibody titer is determined by indirect ELISA.* with non-immune mice
Group compares P < 0.001;P-value is checked according to unpaired t.Square frame represents first and the 3rd quartile;Position in horizontal line representative
Number;Scope must be represented by line.
Figure 45 illustrates the comparison in BALB/c mouse (n=5) and in non-immunity inoculation in the 7th and 25 day after immunity inoculation
The end point titres Log of the IgG antibody produced for pseudomonas aeruginosa strains PAO1 in mice10.Determined anti-by indirect ELISA
Body titre.# is P < 0.0001 compared with non-immune mice group;* P < 0.05 is compared between the 7th and 25 day after immunity inoculation;P-
Value is checked according to unpaired t.Square frame represents first and the 3rd quartile;Horizontal line represents median;Scope must be represented by line.
Figure 46 immunity inoculation is shown after the 34th day BALB/c mouse and the control mice (saline control) of non-immunity inoculation
Produce IgG antibody for multiple pseudomonas aeruginosa strains: AO1, PA28562, PA51430664, PA26132, PAST175,
The cross reactivity (titre) of PA29475 and PA12142.
Figure 47 illustrate by mice the 0th and 15 day with bacterial strain Δ PA4662 preimmunization or the most immune (saline control) it
After, 10 hours BALB/c mouse after infecting with the Pseudomonas aeruginosa wild-type strain PAO1 of the 0.4X dosage of administration in the 22nd day
Liver, spleen and lung in bacterial load (n=8).P-value is checked according to unpaired t.Each point represents the independent of mouse organs
Bacterial load.The meansigma methods often organized is represented by horizontal line.
Figure 48 illustrate respectively with Δ 0380/ Δ 3398, Δ PA4662 and Δ murI/ Δ dat vaccine strains immunity inoculation it
Rear 7th (after the first vaccine dose), 21 (the second dosage), 35 (after the 3rd dosage), 49 (after the 4th dosage) and 63 (
After five dosage, right in BALB/c mouse (the every dosage of n=2-3//every approach) with in non-immunity inoculation of sky in addition to exceptional)
According in mice (saline control) for Acinetobacter bauamnnii strains A TCC 17978 (A), pseudomonas aeruginosa strains PAO1 (B) and
Log Deng the IgG antibody that gene staphylococcus aureus strains 132 Δ spa (C) produces101/ end point titres.Utilization difference is given
Medicine approach intraperitoneal, subcutaneous, intramuscular, intranasal and with different vaccine dose (0.1X and 1X, Δ 0380/ Δ 3398-0.4X
And 0.04X, Δ PA4662 0.2X, 1X, 3X and 10X, Δ murI/ Δ dat) the 0th, 14,28,42 and 56 days (in addition to exceptional)
By mice sequential vaccination.Antibody titer is determined by indirect ELISA.The IgG titre that will produce in the mice of immunity inoculation
Compare with the mice group of non-immunity inoculation.Utilize one way analysis of variance (repeated measure ANOVA) (* P < 0.05;**P<
0.01;* * P < 0.001) determine statistical significance with multiple comparative test (#P < 0.05).Each point represents the independent of mouse IgG
End point titres Log10.The meansigma methods often organized is represented by horizontal line.
Figure 49 immunity inoculation is shown after the 34th day in BALB/c mouse (n=8) and little in the comparison of non-immunity inoculation
The Log of the IgG antibody produced for pseudomonas aeruginosa strains PAO1 in Mus (having intramuscular adminstration)101/ end point titres.Pass through
Indirect ELISA determines antibody titer.* P < 0.0001 compared with control mice group;P-value is checked according to unpaired t.Square frame generation
Table first and the 3rd quartile;Horizontal line represents median;Scope must be represented by line.
Figure 50 is BALB/c mouse after the Pseudomonas aeruginosa PAO1 wild-type strain intra-abdominal infection with 0.4X dosage
Percentage survival (n=8).At the 0th, 14 and 28 days, the mice of immunity inoculation Pseudomonas aeruginosa Δ PA4662 bacterial strain is led to
Cross intramuscular route immunity, and infected with wild-type strain at the 35th day.The 0th, 14 and 28 days little to non-immunity inoculation
Mus is administered saline, and is infecting with wild-type strain on the same day.* the survival of immunity inoculation group compared with the group of non-immunity inoculation
P<0.0001.P-value, checks (log-rank inspection) according to Mantel-Cox.
Figure 51 illustrates over time from 100 μ L Pseudomonas aeruginosa PAO1 wild type and Δ PA4662 bacterial strain (0.4X agent
Amount) quantity (Log of Pseudomonas aeruginosa bacterium colony that recovers of mice (the n=3/ often group) blood inoculated10CFU/mL)。
D-is being supplemented after the drought stress that Figure 52 is shown at 37 DEG C (growth control) at once and 12 and 18 hours
On the TSB flat board of glutamic acid, S. aureus wild type (WT) and double-mutant Δ murI/ Δ dat (Δ Δ) bacterial strain is thin
Born of the same parents' viability.All cultivations and dilution series are carried out the most in triplicate.
Figure 53 is with the survival percentage of BALB/c mouse after staphylococcus aureus 132 wild-type strain intra-abdominal infection
Than (n=5).By mice vaccine serum or inmature serum passive immunity.*, compared with the mice accepting inmature serum, it is administered epidemic disease
The survival P=0.0429 of the mice of Seedling serum.P-value, checks (log-rank inspection) according to Mantel-Cox.
Detailed description of the invention
Definition
In the context of the present invention, term " D-Glu " is interpreted as having molecular formula C5H9NO4, molecular weight 147.129
And there is the compound of D-enantiomeric form or the molecule of glutamic acid (g/mol).Its system entitled " D-Glu ", but it is also
Can with named (but not limited to) " D-Glu ", " D-2-aminovaleric acid ", " glutamic acid D-shaped formula ", " (R)-2-aminovaleric acid " and
H-D-Glu-OH.Its named (2R)-2-aminovaleric acid in IUPAC (IUPAC) system,
And its identifier in " PubChem compound " data base is 6893-26-1.
In the context of the present invention, term " glutamate racemase " is interpreted as being catalyzed Pidolidone to the interconversion reaction of D-
Albumen, it is required for Cell wall synthesis.Its EC identifier (enzyme committee numbering) is 5.1.1.3.This albumen leads to
Crossing its EC coding easily and unchangeably to identify in nucleotide or amino acid sequence database, it refers to that catalysis activity is for Pidolidone
The enzyme of=D-Glu, because its name can change.Therefore, in some Acinetobacter bauamnnii bacterial strains, glutamate racemase
Enzymatic functions can be owing to named ALANINE-LD-glutamate racemase/epimerase, Asp/Glu/ hydantoin
The albumen of racemase, bacitracin synthetase 1 (BA1), aspartic acid/glutamate racemase etc..
In the context of the present invention, term " D-amino acid transaminase " is interpreted as being catalyzed D-alanine and 2-oxoglutaric acid
To acetone acid and the albumen of the interconversion reaction of D-Glu.Its EC identifier is 2.6.1.21.This albumen is encoded by its EC
Easily and unchangeably identifying in nucleotide or amino acid sequence database, it refers to that catalysis activity is for D-alanine+2-ketone penta 2
The enzyme of acid=acetone acid+D-Glu, because its name can change.
In the context of the present invention, term " D-Glu auxotroph " is interpreted as lacking generation D-Glu material
Functional metabolism approach, the antibacterial so specified rely on its growth because this compound can not be synthesized.
In the context of the present invention, term " Murl " is interpreted as and term " glutamate racemase " synonym.
In the context of the present invention, term " Dat " is interpreted as and term " D-amino acid transaminase " synonym.
In the context of the present invention, term " murI " is interpreted as gene or the nucleotide of encoding glutamate racemase protein
Sequence.According to Acinetobacter bauamnnii, Pseudomonas aeruginosa and staphylococcus aureus strains, encoding glutamate racemase protein
Chromogene can be referred to as (but not limited to) murI, murI_1 or murI_2, or they can be by their chromosome
Locus is identified uniquely.
In the context of the present invention, term " dat " is interpreted as gene or the nucleoside of encoding D-amino acid transaminase albumen
Acid sequence.According to staphylococcus aureus strains, the chromogene of encoding D-amino acid transaminase albumen can be referred to as (but not
It is limited to) dat, or it can be identified uniquely by their chromogene seat.
In the context of the present invention, term " inactivates " molecule being interpreted as by one or both of specific gene or albumen
Modification or negative regulation block the expression of specific gene or albumen.Molecular modification includes using conventional recombinant DNA technology, and it is successively
Including: replace one or several nucleotide, insert one or several nucleotide, the partially or completely disappearance of gene, pass through mutation
Chemical induction or radiation-induced destruction.The negative regulation of gene or protein expression includes transcribing and PTGS.
In the context of the present invention, term " Acinetobacter bauamnnii " is defined as belonging to " antibacterial " territory, " mycetozoan
(Proteobacteria) " door, " γ mycetozoan " guiding principle, " pseudomonas (Pseudomonadales) " mesh, " Moraxella
(Moraxellaceae) " section, " acinetobacter calcoaceticus (Acinetobacter) " genus, " calcium acetate/Acinetobacter bauamnnii
(caloaceticus/baumannii) " any microorganism that complex and " Acinetobacter bauamnnii " are planted.So micro-life of definition
Thing is characterised by Gram-negative, strictly aerobic, non-fermented and oxidase negative.
In the context of the present invention, term " ATCC 17978 " refers to have mark in American type culture collection
Know symbol 17978 and belong to " antibacterial " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " Moraxella " section, " no
Lever bacterium " belong to, any bacterial isolates that " calcium acetate/Acinetobacter bauamnnii " complex and " Acinetobacter bauamnnii " are planted.
In the context of the present invention, term " locus A1S_0380 " is defined as Acinetobacter bauamnnii strains A TCC
Physical location " 0380 " in 17978 chromosomes, its gene is referred to as A1S_0380 or murI, and its product is glutamic acid racemization
Pheron.
In the context of the present invention, term " locus A1S_3398 " is defined as Acinetobacter bauamnnii strains A TCC
Physical location " 3398 " in 17978 chromosomes, its gene is referred to as A1S_3398 or murI, and its product is glutamic acid racemization
Pheron.
In the context of the present invention, term " Δ 0380 " is defined as the dye in Acinetobacter bauamnnii strains A TCC17978
Colour solid does not exist locus A1S_0380.
In the context of the present invention, term " Δ 3398 " is defined as the dye in Acinetobacter bauamnnii strains A TCC17978
Colour solid does not exist locus A1S_3398.
In the context of the present invention, term " double sudden change Δ 0380/ Δ 3398 " is defined as at Acinetobacter bauamnnii bacterial strain
There is not locus A1S_0380 and A1S_3398 in the chromosome of ATCC 17978 simultaneously.
In the context of the present invention, term " ATCC 19606 " refers to have mark in American type culture collection
Know symbol 19606 and belong to " antibacterial " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " Moraxella " section, " no
Lever bacterium " belong to, any bacterial isolates that " calcium acetate/Acinetobacter bauamnnii " complex and " Acinetobacter bauamnnii " are planted.
In the context of the present invention, as the term is employed herein " AbH12O-A2 " refers to so name and belong to " thin
Bacterium " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " Moraxella " section, " acinetobacter calcoaceticus " belong to, " calcium acetate/Bao
Graceful acinetobacter calcoaceticus " bacterial isolates planted of complex and " Acinetobacter bauamnnii ".It is quick-fried in the hospital causing several death
The Highly invasive bacterial strain separated in sending out, and it is characterised by that it (is described in Merino about the resistance pattern of Multiple Classes of Antibiotics
etal.,Antimicrob Agents Chemother,54(6):2724-7(2010))。
In the context of the present invention, as the term is employed herein " Ab307-0294 " refers to so name and belong to " thin
Bacterium " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " Moraxella " section, " acinetobacter calcoaceticus " belong to, " calcium acetate/Bao
Graceful acinetobacter calcoaceticus complex " and " Acinetobacter bauamnnii " bacterial isolates of planting.This is Bao Man as pattern pathogen research
The high virulence of acinetobacter calcoaceticus have Capsular strains (such as Russo et al, Infection and Immunity, 78 (9): 3993-
Described in 4000 (2010)).
In the context of the present invention, term " Pseudomonas aeruginosa " be defined as belonging to " antibacterial " territory, " mycetozoan " door,
" γ mycetozoan " guiding principle, " pseudomonas " mesh, " pseudomonas " section, " pseudomonas " belong to any life planted with " Pseudomonas aeruginosa "
Object.Pseudomonas aeruginosa is to have the Gram-negative of one pole mobility, aerobic, coccobacillus antibacterial.
In the context of the present invention, term " PAO1 " refers to have general identifier PAO1 and belong to " antibacterial " territory, " become
Shape bacterium " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " pseudomonas " section, " pseudomonas " belong to and " Pseudomonas aeruginosa " plant
Any bacterial isolates.
In the context of the present invention, the thing during term " PA4662 " is defined as pseudomonas aeruginosa strains PAO1 chromosome
Reason position " 4662 ", its gene is referred to as PA4662 or murI, and its product is glutamate racemase albumen.
In the context of the present invention, term " Δ PA4662 " is defined as the chromosome at pseudomonas aeruginosa strains PAO1
In there is not locus PA4662.
In the context of the present invention, as used herein, term " PA28562 ", " PA51430664 ", " PA26132 " and
" PA29475 " refers to so name and belong to " antibacterial " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " vacation
Zymomonas mobilis " section, " pseudomonas " belong to and " Pseudomonas aeruginosa " bacterial isolates of planting.
In the context of the present invention, as used herein, term " PAST175 " refers to have MLST sequence type 175
And belong to " antibacterial " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas " mesh, " pseudomonas " section, " false (ST175)
Zymomonas mobilis " belong to the bacterial isolates planted with " Pseudomonas aeruginosa ".Such as M.Garc í a-Castillo et al, Journal of
Described in Clinical Microbiology 49 (2011) 2905-2910, this bacterial strain was accredited as the height of international communication in the past
Risk is cloned.
In the context of the present invention, as used herein, term " PA12142 " refers to such as M.Tom á s et al,
Bacterial isolates 12142 described in Antimicrobial Agents and Chemotherapy 54 (2010) 2219-2224
(popular Liverpool bacterial strain).This bacterial strain belongs to " antibacterial " territory, " mycetozoan " door, " γ mycetozoan " guiding principle, " pseudomonas "
Mesh, " pseudomonas " section, " pseudomonas " belong to and " Pseudomonas aeruginosa ".
In the context of the present invention, term " staphylococcus aureus " is defined as belonging to " antibacterial " territory, " heavy wall bacterium
(Firmicutes) " door, " bacillus cereus (Bacilli) " guiding principle, " bacillus cereus (Bacillales) " mesh, " staphylococcus
(Staphylococcaceae) " section, " staphylococcus (Staphylococcus) " belong to the micro-life planted with " staphylococcus aureus "
Thing.So microorganism of definition is characterised by Gram-positive, amphimicrobian, coccus antibacterial.
In the context of the present invention, term " 132 " refers to have same names and belong to " antibacterial " territory, " heavy wall bacterium "
Door, " bacillus cereus " guiding principle, " bacillus cereus " mesh, " staphylococcus " section, " staphylococcus " genus are planted with " staphylococcus aureus "
Any bacterial isolates.This is clinical methicillin resistant strain (Vergara-Irigaray et al, Infection and
Immunity, 77 (9): 3978-3991 (2009)), and it is used as model in this invention to produce " Staphylococcus aureus
Bacterium " Auxotrophic mutant.
In the context of the present invention, term " Δ murI " is defined as the chromosome at staphylococcus aureus strains 132
In there is not locus murI.
In the context of the present invention, term " Δ dat " is defined as in the chromosome of staphylococcus aureus strains 132
There is not locus dat.
In the context of the present invention, term " double sudden change Δ murI/ Δ dat " is defined as at staphylococcus aureus strains
There is not murI and dat gene in the chromosome of 132 simultaneously.
In the context of the present invention, as used herein, term " 132 Δ spa " represents have same names and belong to
" antibacterial " territory, " heavy wall bacterium " door, " bacillus cereus " guiding principle, " bacillus cereus " mesh, " staphylococcus " section, " staphylococcus " belong to and " gold
Staphylococcus aureus " microorganism planted.So the microorganism of definition is the staphylococcus aureus in spa gene with disappearance
132 bacterial strains (Vergara-Irigaray et al, Infection and Immunity, 77 (9): 3978-3991 (2009)).
In the context of the present invention, as used herein, term " RN4220 " represents have same names and belong to " thin
Bacterium " territory, " heavy wall bacterium " door, " bacillus cereus " guiding principle, " bacillus cereus " mesh, " staphylococcus " section, " staphylococcus " belong to and " golden yellow
Staphylococcus " microorganism planted.So definition microorganism be staphylococcus aureus clone intermediate strains (Nair et al,
Journal Bacteriology,193(9):2332-2335(2011))。
In the context of the present invention, as used herein, term " USA300LAC " represents have same names and belong to
" antibacterial " territory, " heavy wall bacterium " door, " bacillus cereus " guiding principle, " bacillus cereus " mesh, " staphylococcus " section, " staphylococcus " belong to and " gold
Staphylococcus aureus " microorganism planted.So the microorganism of definition is the stream causing the Community Acquired Infections in healthy individuals
Row methicillin resistant S aureus strains (Diep et al, Lancet 4;397(9512):731-739
(2006))。
In the context of the present invention, as used herein, term " RF122 " refers to have MLST sequence type 151 (ST175)
With clone complex 151 (CC151) and belong to " antibacterial " territory, " heavy wall bacterium " door, " bacillus cereus " guiding principle, " bacillus cereus " mesh,
" staphylococcus " section, the bacterial isolates that " staphylococcus " belongs to and " staphylococcus aureus " is planted.So microorganism of definition is point
From certainly present clinical mastitis milch cow cause bovine mastitis bacterial strain (Herron-Olson L et al, PloS ONE2:
e1120(2007))。
In the context of the present invention, as used herein, term " ED133 " refers to have MLST sequence type 133 (ST175)
With clone complex 133 (CC133) and belong to " antibacterial " territory, " heavy wall bacterium " door, " bacillus cereus " guiding principle, " bacillus cereus " mesh,
" staphylococcus " section, the bacterial isolates that " staphylococcus " belongs to and " staphylococcus aureus " is planted.So definition microorganism be
The bacterial strain (Guinane et al, Genome Biol Evol 2:454-466 (2010)) causing bovine mastitis that France separates.
In the context of the present invention, as used herein, term " ED98 " refer to have MLST sequence type 5 (ST175) and
Clone complex 5 (CC5) and belong to " antibacterial " territory, " heavy wall bacterium " door, " bacillus cereus " guiding principle, " bacillus cereus " mesh, " Fructus Vitis viniferae ball
Bacterium " section, " staphylococcus " belong to and " staphylococcus aureus " bacterial isolates of planting.So definition microorganism be isolatable from ill
Broiler chicken fowl adapt to bacterial strain (Lowder et al, PNAS 106 (46): 19545-50 (2009)).
In the context of the present invention, term " bacterial isolates of glutamate racemase defect " is interpreted as producing function
And/or any bacterial isolates of the glutamate racemase of activity form.This defect can be due to: blocks its encoding gene
Express, affect posttranscriptional modification and the post translational modification of enzymatic activity, the allosteric regulation of this enzyme or cell location.
In the context of the present invention, term " passive immunity " is used to refer to be administered to this individuality to give individual immunity
Antibody or its fragment.
In the context of the present invention, express " therapeutically effective amount " refer to allow produce desired effects the present invention antibody or
The amount of the attenuated bacteria bacterial strain of the present invention.The pharmaceutically acceptable adjuvant and the carrier that may be used for described compositions are this area skills
Carrier known to art personnel.The compositions that the present invention provides can be promoted by any route of administration, for this purpose may be used
With described compositions is formulated as suitable dosage form and with for selected route of administration at pharmacologically acceptable excipient
Prepare together.
In the context of the present invention, term " vaccine " refers to for setting up the immune antigen system to disease response
Product.
Detailed Description Of The Invention
As it has been described above, if bacterial isolates has completely lost the ability producing D-Glu, then it is D-Glu nutrition
Deficiency.In the present invention, we should show that the auxotrophic bacterial isolates of D-Glu has so that they are special
It is suitable as the feature of vaccine.So situation is exactly, because these certain types of bacterial isolates (D-Glu auxotrophies
Type) the most nontoxic (attenuation) to avoid unacceptable pathologic effect, host induces the immunity of enough levels, and substantially
On can not recover to poisonous wild-type strain.
All these it is characterized as that design and preparation have the vaccine of potential application (universality) in various bacterial isolateses and carry
Supply new technology platform.This true (versatility of the present invention or universality) clearly confirms that in the present invention, because author
By the present invention at 3 completely irrelevant bacterial species (Acinetobacter bauamnnii (A.baumannii), Pseudomonas aeruginosa
(P.aeruginosa) and staphylococcus aureus (S.aureus)) in put into practice.For the antibacterial thing that these are completely irrelevant
Each of in kind, prepare D-Glu auxotrophic strain and as Examples below confirms, all these mutant bacterias
Strain the most nontoxic (attenuation) is to avoid unacceptable pathologic effect, but can induce exempting from of enough levels in host
Epidemic disease.The all these bacterial strain of result is all outstanding vaccine candidate object.
In order to confirm above, the author of the present invention is firstly the need of proving that all auxotroph D-Glu bacterial strains cause not
The attenuated strain of unacceptable pathologic effect can be produced in host.For this purpose, author uses nosocomial pathogens Bao
Graceful acinetobacter calcoaceticus (sees embodiment 5).
For this purpose and as described in Example 5, the author of the present invention is on the one hand by intraperitoneal administration Bao Man not
The inoculum of lever bacterium wild-type strain ATCC 17978, and on the other hand by the double mutant strain Δ of intraperitoneal administration
0380/ Δ 3398 Acinetobacter bauamnnii of the enzyme glutamate racemase defect (and be therefore D-Glu auxotrophic strain)
Inoculum carrys out inducing systemic sexuality dye in BALB/c mouse.
Figure 10 A illustrates the partial results tested from this;Particularly when infecting with the wild-type strain increasing dosage
The different weight percentage of mouse survival.From these data it may be concluded that the dosage from 2X provides gradually subtracting of mouse survival
Few.It is in fact possible to observe the fatal dose 100 (LD of wild-type strain ATCC17978100) (be interpreted as subtracting mouse survival
Few to minimum dose necessary to 0%) it is 2.5X.Additionally, this figure also shows when dosage is at more than 3X, it was observed that survival hundred
Proportion by subtraction quickly reduces.
On the other hand, Figure 10 B illustrates that the mice with double mutant strain Δ 0380/ Δs 3398 infection increasing dosage is not
The survival rate of same level.In this case, and substantially contrast with Figure 10 A, LD100(lethal 100) are 6X.This dosage is worked as
More much higher than the fatal dose corresponding to wild-type strain.It is true that 6X dosage is the highest, so that the author of the present invention is hard
The death of letter mice is not as the duplication of antibacterial, and is because septic shock, and this clearly shows this (double mutant strain)
It it is nontoxic or the bacterial isolates of attenuation.
In addition and in order to confirm the above results, can observe as in embodiment 6, the author of the present invention is given by intraperitoneal
The following inoculum of medicine Acinetobacter bauamnnii: wild-type strain ATCC 17978 and mutant strain Δ 0380, Δ 3398 and Δ
0380/ Δ 3398 is inducing systemic sexuality dye in BALB/c mouse.
In this sense, it is noted that following critically important, as described in embodiments of the invention, previously paragraph makes
With with in all bacterial strains mentioned, it is only considered that double mutant strain Δ 0380/ Δ 3398 is that D-Glu is auxotrophic, therefore
Need to add external source D-Glu in the medium for growing and surviving.Additionally, be also noted that embodiment 6 explanation acute
In the model infected, infect and occur, by the blood fast propagation of antibacterial, within 11-30 hour, cause mice dead the most after infection
Die (see Figure 10 A).End, it is noted that to following the most critically important, the counting of antibacterial from liver, the author of the present invention can obtain difference
The aggressive of bacterial strain and the measurement of replication capacity.
Describing in Figure 11 and show, following meansigma methods is derived from embodiment 6 experiment of explanation: using wild-type strain sense
8.29Log in the liver of those mices of dye10CFU (colony-forming units)/g;Those mices with Δ 0380 strain infection
6.88Log in liver10CFU/g;8.06Log in the liver of those mices infected with mutant strain Δ 339810CFU/g;And
With 1.59Log in the liver of those mices of double mutant strain Δ 0380/ Δs 3398 infection10CFU/g。
Based on these as a result, it is possible to reach a conclusion, double mutant strain Δ 0380/ Δs 3398 and the meter of mutant strain Δ 0380
Number is substantially less than the counting observing wild-type strain.The most noticeable and obvious minimizing is to analyze double mutant strain Δ
The minimizing observed during the colony counting of 0380/ Δ 3398, wherein obtains the almost 7Log of average bacteriological load10The minimizing of value.
Additionally, surprisingly, in 44.4% mice infected with double mutant strains, do not recover antibacterial.
As a result, this clearly shows that this (double mutant strain) is nontoxic or attenuated bacteria bacterial strain again.
While it is true, the author of the present invention is in order to confirm the universality of platform, with extra and completely irrelevant about two kinds
The auxotroph D-Glu bacterial strain of bacterial species, i.e. Pseudomonas aeruginosa and staphylococcus aureus carries out further reality
Test.As we hereinafter it should be observed that, the auxotrophic strain of both species also produces and can not produce in host
The attenuated strain of unacceptable pathologic effect.
In this sense, as described in embodiment 16, in order to determine acute septicopyemia infect during the cause of these bacterial strains
The purpose of dead dosage, is administered Pseudomonas aeruginosa PAO1 wild-type strain and the mutant strain Δ of various dose to BALB/c mouse
PA4662.After infecting, monitor mice 7 days, and the bacterial infection of various dose is determined survival rate.
Figure 24 A we can observe that with moving that the Pseudomonas aeruginosa PAO1 wild-type strain increasing dosage infects
Survival in various degree in thing.For this bacterial strain, LD100For=0.4X.Figure 24 B we can observe that and use dose
Survival in various degree in the animal that the Δ PA4662 mutant strain of amount infects.For this bacterial strain, LD100For > 40X, can lead
Cause the bacterial inoculum that mice dies from the very high dose of septic shock (and being not due to antibacterial duplication).This shows with wild
Raw type bacterial strain is compared, and this bacterial strain (Δ PA4662) has the virulence (LD than wild-type strain greatly reduced100High 100 times
Dosage only reduces by 50% mouse survival rate).
Finally, the author of the present invention is by using another bacterial species to be further characterized by platform technology pervasive of the present invention
Property concept, i.e. by prove also to produce about the auxotroph D-Glu bacterial strain of species staphylococcus aureus can not be in place
Master produces the attenuated strain of unacceptable pathologic effect.
In this sense, and as described in embodiment 25, by coming with 3% pig mucin intraperitoneal injection
BALB/c mouse produces systemic infection with staphylococcus aureus 132 wild type and double mutant strain.
As shown in Figure 35 A, determine that the minimum dose that the survival of these mices reduces the wild-type strain to 0% is LD100
=3X.Substantially contrasting with Figure 35 A, Figure 35 B explanation inoculation is than the LD of wild-type strain100The double-mutant of high 10-multiple dose causes
100% survival rate.Therefore, the fatal dose of double-mutant is more than 30X LD100>30X.This clearly confirms that staphylococcus aureus
Double-mutant be the attenuated strain showing the virulence potentiality lower than wild type counterparts.
Generally speaking, these data confirm that the auxotrophic all bacterial isolateses of D-Glu are all attenuations.But,
It is known that bacterial isolates attenuation is insufficiently to serve as vaccine candidate object, because it must also be able to produce immunne response and lure
Lead protection.Additionally, these mutant strains should preferably comprise gives all antigens that cross protection is required in immunity.
Therefore, in order to evaluate the antibody-mediated immunne response to different immunization scheme, author is carried out example 8
The experiment of middle explanation, wherein by utilizing double mutant strain Δ 0380/ Δ 3398 intraperitoneal injections of 1X dosage at the 0th and 14 day
By BALB/c mouse immunity.Additionally, be administered saline serum at the 0th and 14 day in the same manner to another group control mice.In immunity
After inoculation the 7th day, fetch the serum of the mice of vaccine (being administered at the 0th day) immunity of single dose of using by oneself, similarly,
After being administered for the first time 21 days, also fetch from also with the serum of the mice of doses remaining immunity, together with saline serum of using by oneself
The serum of the control mice of injection.
These serum is used for by utilizing elisa technique to determine for including ATCC 17978, ATCC 19606 and
AbH12O-A2 is in the titre of the antibody (IgG) of the different strains of interior Acinetobacter bauamnnii, and it is wide therefore to measure vaccine generation
The ability (embodiment 9) of general immunne response.It should be noted that Acinetobacter bauamnnii strains A bH12O-A2 is Highly invasive
Bacterial strain, separates in the hospital killing numerical digit patient is broken out, and is characterised by the resistance pattern to Multiple Classes of Antibiotics.
In the mice of all immunity, compared with the mice of non-immunity inoculation, detect for wild-type strain is notable
The antibody (Figure 13) of level.Compared with the mice of non-immunity inoculation, antibody produces and significantly raised at the 7th and 21 day, exists the most respectively
After injecting double mutant bacterial strain Δ 0380/ Δs 3398 of the first dosage and the second dosage.Additionally, at the 21st day, (2 times continuously
After double mutant strain Δ 0380/ Δ 3398 of injection) generation of antibody is significantly higher than the generation of the 7th day antibody.Obtained at the 21st day
The antibody for Acinetobacter bauamnnii wild-type strain ATCC 17978 generation with for Acinetobacter bauamnnii bacterial strain
Those similar (Figure 15) that ATCC 19606 and Acinetobacter bauamnnii AbH12O-A2 obtains.This result confirms to use bacterial strain Δ
0380/ Δ 3398 immunity inoculation not only produces the antibody for wild-type strain, but also produce for have different resistance with
Other bacterial isolateses such as ATCC 19606 bacterial strain of virulence pattern and the IgG antibody of strains A bH12O-A2 reaction.
Additionally, in order to prove that these antibacterials (the auxotrophic bacterial isolates of D-Glu) can produce and exempt from by force further
Epidemic disease response, is further tested in bacterial species Pseudomonas aeruginosa and staphylococcus aureus.
As described in embodiment 16, the 0.1X Pseudomonas aeruginosa Δ PA4662 mutant strain of a vaccine dose be enough to
The notable IgG of triggering produces, even if within the 40th day, being also so (Figure 44) after administration when detecting.But, equal to or more than 0.4X
Vaccine dose cause the IgG of higher level to produce.Additionally, IgG produces significantly higher (figure when being administered the 2nd vaccine dose
45)。
Additionally, about the different strains of Pseudomonas aeruginosa, be derived from the mice by Δ PA4662 immunity inoculation and to
The serum of the mice of medicine saline carries out ELISA and produces the ability of extensive immunne response to measure Δ PA4662 vaccine.Use bacterial strain
PA28562 obtains and those the similar results observed about pseudomonas aeruginosa strains PAO1, but about remaining survey
The pseudomonas aeruginosa strains of examination also sees that high-caliber cross reactivity (Figure 46).This confirmation bacterial strain Δ PA4662 immunity connects
Kind not only produces for the antibody waiting gene wild-type strain, but also what generation was reacted for multiple pseudomonas aeruginosa strains
IgG antibody.
Finally, as described in embodiment 28 and 29, the author assessment of the present invention antibody-mediated for golden yellow Portugal
The immunne response of grape meningitidis strains (referred to as staphylococcus aureus 132 Δ spa, A protein deficiency) and coming for humans and animals
The cross reactivity of 4 kinds of unrelated staphylococcus aureus strains in source.For this purpose, abdominal cavity was passed through at the 0th and 14 day
Interior injection double-mutant Δ murI/ Δ dat carrys out immune mouse.The blood serum sample from every mice collected at the 21st day is used
Determine for waiting gene staphylococcus aureus 132 Δ spa bacterial strain (Figure 39) and for popular MRSA Community-acquired
The titre of the antibody (IgG) of 3 kinds of bacterial strains (Figure 40) of USA300LAC bacterial strain and animal origin.As shown in figures 39 and 40, with rather
Epidemic disease inoculation mice compare, with double-mutant Δ murI/ Δ dat bacterial strain immunity all mices produce significant level for often
Plant the specific antibody of antibacterial, it was demonstrated that staphylococcus aureus Δ murI/ Δ dat mutant causes the ability of IgG antibody, described
The gene Δ spa homologues such as IgG antibody not only identification, but also identify clinical epidemic strains and be isolatable from different hosts such as
Other bacterial strains of cattle, sheep and poultry.
Therefore, the data up to the present shown not only confirm that the auxotrophic all bacterial isolateses of D-Glu are to subtract
Poison, produce immunne response but it also turn out that these bacterial strains comprise and give all antigens that cross protection is required in immunity.
Finally, in order to verify whether the auxotrophic bacterial isolates of D-Glu gives the protection of acceptable level, this
The author of invention carries out series of experiments to evaluate double mutant strain Δ 0380/ Δ 3398 with nosocomial pathogens Acinetobacter bauamnnii
Effectiveness as vaccine.As described in embodiment 10, it was administered mutant strain Δ at the 0th and 14 day to BALB/c mouse
0380/Δ3398.Saline it is administered the most in the same manner the 0th and 14 day control mice.After injection 21 days for the first time, motionless with Bao Man
Bacillus strain ATCC17978, AbH12O-A and Ab307-0294 attack mice independently to set up lethal systemic infection.Attack
After hitting, monitoring mice 7 days to determine compared with control mice (non-immunity inoculation), the survival rate of immunized mice.
When with Acinetobacter bauamnnii ATCC 17978 strain infection, first 24 hours periods are in the mice group of non-immunity inoculation
In observe 11 death, this represent in this group mortality rate of 92%.By contrast, the mice of all immunity inoculations is all attacked against each other
Hitting survival, overcome infection, this represents 100% survival (see Figure 16) rate in this group.
Additionally, determine by whether the response produced with Δ 0380/ Δ 3398 bacterial strain immunity be enough to provide for other
The protection of Acinetobacter bauamnnii bacterial strain lethal infection, including high virulence and pathogenic bacterial strains.With AbH12O-A2 strain challenge
In the case of, first 19 hours periods observed 9 death in the mice group of non-immunity inoculation, and this represents the mortality rate of 100%.Phase
Under Bi, the mouse survival (100% survival rate) (see Figure 17) of all immunity inoculations.
In the case of have Capsular strains to attack with Ab307-0294, in first 24 hours, we have recorded non-immunity inoculation
Mice group in 100% mortality rate and in the past by 83% survival rate in the mice group of Δ 0380/ Δ 3398 bacterial strain immunity
(see Figure 18).This confirms that the Acinetobacter bauamnnii bacterial strain that can give for having notable virulence by mutant strain immunity inoculation is drawn
The protection of the systemic infection risen.
All these results show can provide for infecting different groups by Δ 0380/ Δ 3398 bacterial strain immunity inoculation
The protective immunity of Acinetobacter bauamnnii bacterial strain.
Additionally, be also whether D-Glu other bacterial isolateses auxotrophic give acceptable level to verify
Protection, the author of the present invention with pathogen Pseudomonas aeruginosa (as described in embodiment 18) and staphylococcus aureus (as
Explanation in embodiment 26 and 27) carry out extra experiment.
As described in embodiment 18, it was administered mutant strain PA4662 at the 0th and 14 day to BALB/c mouse.Identical
Natural law is only administered saline to control mice.After injection 25 days for the first time, attack mice with pseudomonas aeruginosa strains PAO1, with
Just lethal systemic infection is set up.After attack, monitoring mice 7 days to determine compared with control mice, immunized mice
Survival rate.
When infecting with Pseudomonas aeruginosa PAO1 wild-type strain, first 15 hours periods are in the mice group of non-immunity inoculation
In observe 8 death, this represent in this group mortality rate of 100%.By contrast, the mice of all immunity inoculations is all attacked against each other
Hitting survival, overcome infection, this represents 100% survival (see Figure 26) rate in this group.
As described in embodiment 26, in order to evaluate staphylococcus aureus double sudden change Δ murI/ Δ dat bacterial strain as epidemic disease
The effectiveness (level of protection) of Seedling, at the 0th and 14 day by BALB/c mouse double-mutant Δ murI/ Δ dat intraperitoneal immunity.
It is administered saline to one group of mice at identical natural law.At the 21st day, with the lethal inoculation of S. aureus wild type bacterial strain
Thing infecting mouse.20 or 22 hours after infection (respectively Figure 36 and 37), determines the count of bacteria in mice spleen and blood.This
Sample is it was confirmed by the protective effect of the double-mutant Δ murI/ Δ dat immunity inoculation of staphylococcus aureus, because with this
The inoculation of bacterial strain preimmunization causes substantially reducing of bacterial load.
Additionally, as shown in embodiment 27, when with the staphylococcus aureus 132 wild-type strain attack Δ of fatal dose
During the BALB/c mouse of murI/ Δ dat bacterial strain preimmunization, when compared with non-immune mice, observe the significance difference of survival
Different.In this case, 8 mices of immunity inoculation group, to attacking survival, overcome infection, and this represents 61.5% deposits in this group
Motility rate (Figure 38), and in not immunity inoculation group, observe the mortality rate of 90%.
Generally speaking, these results confirmation Δ PA4662 and Δ murI/ Δ dat bacterial strain immunity inoculation provide respectively for
Infect Pseudomonas aeruginosa and the protective immunity of staphylococcus aureus.
EXPERIMENTAL EXAMPLE as herein described provides method and result, and it confirms the auxotrophic bacterial isolates of D-Glu
The most nontoxic (attenuation), to avoid unacceptable pathologic effect, induces exempting from of enough levels independent of route of administration in host
Epidemic disease, and for they uses in actively or passively immunity, there is basic level of security (environment and for host).
Therefore, an embodiment of the present invention relates to being no longer able to producing the vaccine candidate object that is suitable as of D-Glu
Living toxic-reduced bacteria.
The living toxic-reduced bacteria being used as vaccine candidate object according to the present invention can be obtained by several ways as explained below.
In this sense, understanding following critically important, Gram-positive and gram negative bacteria are respectively provided with peptidoglycan cell wall, and peptide gathers
Sugar cell wall gives they character shapes and provides mechanical protection for them.The also referred to as Peptidoglycan of murein is by sugar and amino
The polymer of acid composition, it reticulates layer in the plasma membrane profile of antibacterial (but not being archeobacteria), forms cell wall.Saccharic composition by β-
(1,4) N-acetyl-glucosamine connected and the alternate residues composition of-acetylmuramic acid.
Peptidoglycan layer substantially ((20-80 nanometer)) ratio (7-8 in gram negative bacteria in gram-positive bacterium
Nanometer) thick, connect S-layer.Peptidoglycan forms about the 90% of gram-positive bacterium dry weight, but only gram negative strain
10%.Therefore, the existence of high-caliber Peptidoglycan is that antibacterial is characterized as gram-positive main determining factor.At gram sun
In property bacterial strain, it is critically important in adhesive attraction and serotype purpose.For Gram-positive and gram negative bacteria, about
The granule of 2nm can pass through peptidoglycan cell wall.
As described in clear in Fig. 1 and 2, D-Glu is one of key component of peptidoglycan cell wall, therefore D-paddy ammonia
Acid is required for whole cell peptidoglycan synthesis.The formation (see Fig. 3) of two kinds of enzyme catalysis D-Glus:
1. glutamate racemase (EC 5.1.1.3), Murl, a kind of enzyme being catalyzed following chemical reaction
PidolidoneD-Glu
Therefore, this enzyme has a kind of substrate Pidolidone and a kind of product D-Glu;And
2.D-amino acid transaminase, Dat (EC 2.6.1.21), a kind of enzyme being catalyzed following chemical reaction:
D-alanine+2-oxoglutaric acidAcetone acid+D-Glu
Therefore, two kinds of substrates of this enzyme are D-alanine and 2-oxoglutaric acid, and its two kinds of products are acetone acid and D-
Glutamic acid.
Once synthesize, D-Glu as monomeric unit by enzyme-specific, i.e. by enzyme UDP-N-acetylmuramyl-
ALANINE: D-Glu ligase (EC 6.3.2.9) adds to peptidoglycan cell wall.
Therefore, it is possible to two kinds of enzymes that catalysis D-Glu is formed are glutamate racemase and D-amino acid transaminase.But,
Enzyme D-amino acid transaminase is not present in all bacterial isolateses.By contrast, glutamate racemase gene gathers at generation peptide
All species of sugar are guarded, such as Acinetobacter bauamnnii, anthrax bacillus, bacillus cereus, bacillus subtilis, excrement intestinal ball
Bacterium, enterococcus faecalis, escherichia coli, helicobacter pylori, Klebsiella Pneumoniae, staphylococcus aureus, Pseudomonas aeruginosa, change
Streptococcus pyogenes etc..
In these antibacterials of the many including escherichia coli, therefore there is the unique egg that can be catalyzed D-Glu synthesis
In vain, i.e. glutamate racemase.In these cases, this enzyme is requisite for bacterial growth, because it is in cell
The exclusive source of D-Glu.But, at other antibacterials such as staphylococcus haemolyticus, Bacillus sphaericus (Bacillus
Sphaericus), in bacillus cereus (Bacillus sp.) YM-1 and staphylococcus aureus, Dat and Murl albumen all exists,
Therefore can have complementary functions mutually.
Such as escherichia coli, Acinetobacter bauamnnii ATCC 17978 does not has Dat albumen, but comprises two of coding MurI not
Homogenic, particularly MurI 1 (locus A1S_0380) and MurI 2 (A1S_3398 locus).Two genes are at aminoacid
Level shows mutually 29.8% homogeny, and goes out 33.2 and 23.2% phase respectively with e. coli k12 MurI protein expression
The same sex.
Therefore, D-Glu auxotrophic strain can be by the gene or not of inactivation encoding glutamate racemase MurI
Homogenic it is readily available, for comprising those antibacterials of Dat and MurI albumen further, also has and inactivate encoding D-ammonia extraly
The gene of base acid transaminase or multiple gene.This represents for those antibacterials without Dat enzyme, in some cases, in order to produce
Raw D-Glu auxotrophic strain, needs to inactivate the term single gene of glutamate racemase, as at coli strain K12 and
In the case of pseudomonas aeruginosa strains PAO1, in other cases, need to inactivate two differences of encoding glutamate racemase
Gene, as in the case of Acinetobacter bauamnnii ATCC 17978 bacterial strain, or in some other cases, needs to inactivate paddy ammonia
3 different genes of acid racemase, as in the case of Acinetobacter bauamnnii strains A BNIH10, i.e. encode paddy ammonia by inactivation
The full gene of acid racemase.In other cases, need to inactivate gene and the codase D-aminoacid of encoding proteins MurI
Transaminase, those genes of Dat, as some gram-positive bacteriums such as staphylococcus aureus, staphylococcus haemolyticus, spherical
In the case of bacillus cereus and bacillus cereus YM-1.
As a result, a first aspect of the present invention relates to a kind of for designing and producing based on the auxotrophic work of D-Glu
The new platform of attenuated bacteria bacterial strain and technology.This new platform technology has potentiality (universality) in various bacterial isolateses.This
The individual fact (versatility of the present invention or universality) clearly confirms that the most, because as shown in the Examples, author will
The present invention is at 3 completely irrelevant bacterial species (Acinetobacter bauamnnii (A.baumannii), Pseudomonas aeruginosa
(P.aeruginosa) and staphylococcus aureus (S.aureus)) in put into practice.
Therefore, a preferred embodiment of a first aspect of the present invention relates to one and prepares and comprise D-Glu auxotrophy
Type mutant is lived the method for pharmaceutical composition (preferred vaccine) of bacterial isolates, wherein said pharmaceutical composition be suitable for animal and/
Or people (infects it for the prophylactic treatment of the infection of the wild-type form of the Auxotrophic mutant bacterial isolates of compositions
Before) and/or therapeutic treatment (after the Disease Clinical performance after infection or caused by infection), and wherein said medicine group
Compound is prepared by the method comprised the following steps:
A. D-Glu Auxotrophic mutant bacterial isolates alive is obtained;
B. bacterial isolates of being lived by described Auxotrophic mutant joins pharmaceutically acceptable carrier or diluent and appoints
Selection of land adds adjuvant;And
The most optionally pharmaceutical composition described in lyophilization.
In another preferred embodiment of a first aspect of the present invention, described preparation method comprises the following steps:
A. provide and can express glutamate racemase and possible D-amino acid transaminase and comprise peptidoglycan cell wall
Bacterial isolates;
B. it is no longer able to expressive function glutamate racemase and/or functional D-amino acid transaminase with bacterial isolates
Mode inactivates the gene of encoding glutamate racemase or multiple gene, and if it is required, inactivation codase D-aminoacid turns ammonia
The gene of enzyme or multiple gene, therefore the inactivation of wherein said gene causes described bacterial isolates to become D-Glu auxotrophy
Type;And
C. bacterial isolates of being lived by described Auxotrophic mutant joins pharmaceutically acceptable carrier or diluent and appoints
Selection of land adds adjuvant;And
The most optionally pharmaceutical composition described in lyophilization.
The inactivation mentioned in above step b) can be to insert, lack, replace or combinations thereof, as long as described inactivation is led
Cause can not expressive function glutamate racemase and/or functional D-amino acid transaminase albumen.Functional glutamate racemase
And/or functional D-amino acid transaminase albumen is interpreted as the albumen with the regulation feature of wild-type protein.It is therefore contemplated that lack
Fall into and therefore can not participate in D-Glu synthesize glutamate racemase and/or functional D-amino acid transaminase albumen be NOT function
Can property albumen.
In another preferred embodiment or its any preferred embodiment of a first aspect of the present invention, described medicine group
Compound is vaccine, and described preparation method includes adding adjuvant.
In another preferred embodiment or its any preferred embodiment of a first aspect of the present invention, step a's) is thin
Bacteria strain is Gram-positive or gram negative bacteria.Preferably, the bacterial isolates of step a) is thin selected from consist of
Fungus kind list: Acinetobacter bauamnnii, Acinetobacterbaylyi, acinetobacter calcoaceticus, acinetobacter haemolyticus, Qiong Shi
Acinetobacter calcoaceticus, acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacter pittii, radioprotective
Acinetobacter calcoaceticus, actinobacillus lignieresii, actinobacillus suis, Aeromonas caviae, Aeromonas hydrophila, Aeromonas veronii
Subsp.Sobria, actinobacillus actinomycetem comitans, Arcobacter butzleri, Arcobacter nitrofigilis, Xie Dian
Afnyloliquefaciens, anthrax bacillus, Bacillus bataviensis, solution fiber bacillus cereus, bacillus cereus, gram Lloyd's's bud
Spore bacillus, Bacillus licheniformis, bacillus megaterium, Bacillus pumilus, bacillus subtilis, bacillus thuringiensis, crisp
Weak bacteroid, Bordetella avium, bordetella branchiseptica, Bordetella pertussis, Bordetella petrii,
Bacillus abortus, brucella melitensis, Brucella suis, Burkholderia cepacia, glanders burkholderia,
Burkholderia multivorans, Burkholderia Pseudomallei, Burkholderia thailandensis, the most curved
Aspergillosis, campylobacter fetus fetus subspecies, campylobacter fetus sexually transmitted disease (STD) subspecies, very thin Campylobacter spp, Campylobacter hominis,
Campylobacter jejuni, rectum Campylobacter spp, Showa Campylobacter spp, Uppsala Campylobacter, citrobacter freundii, kirschner citric acid bar
Bacterium, Clostridium asparagiforme, bacillus botulinus, Bacillus butyricus, clostridium difficile, aerogenesis
Perfringens bacillus, Clostridium saccharobutylicum, Clostridium tetani, Corynebacterium diphtheriae bar
Bacterium, Corynebacterium pseudotuberculosis, clostridium perfringen, enterobacter cloacae, enterococcus faecalis, enterococcus faecalis, erysipelothrix ruhsiopathiae, large intestine bar
Bacterium, actinomyces pseudonecrophorus, fusobacterium nucleatum, adjacent particles chain bacterium, severe foster particle chain bacterium, horse reproductive tract haemophilus, influenza are bloodthirsty
Bacillus, haemophilus parainfluenzae, haemophilus paragallinarum, haemophilus parasuis, pleuropneumonia haemophilus, Haemophilus somnus,
Helicobacter pylori, Klebsiella oxytoca, Klebsiella Pneumoniae, Oak Ridge legionella, legionella pneumophilia, hyperbolic hook end spiral
Body, leptospira illini, leptospria interrogans, Listeria monoeytogenes, capitate lysine bacillus cereus, spherical
Lysine bacillus cereus, cattle catarrhalis, morganella morganii, mycobacterium abscessus, mycobacterium africanum, Mycobacterium avium, cattle are divided
Branch bacillus, Mycobacterium leprae, mycobacterium tuberculosis, gonococcus, Neisseria meningitidis, pasteurella multocida,
Plesiomonas shigelloides, propionibacterium acnes, Proteus hanseri, proteus mirabilis, Pseudomonas aeruginosa, hog cholera
Salmonella, Salmonella enteritidis intestinal subspecies, Salmonella enteritidis, salmonella paratyphi, salmonella typhi, general city sand thunder
Bacterium, shigella boydii, Shigella dysenteriae, shigella flexneri), A Erlaite staphylococcus, staphylococcus aureus, head Fructus Vitis viniferae
Coccus, Staphylococcus caprae, Staphylococcus carnosus, staphylococcus epidermidis, Staphylococcus equorum, staphylococcus haemolyticus, people's Fructus Vitis viniferae ball
Bacterium, road Deng's staphylococcus, Staphylococcus pasteuri, Staphylococcus pettenkoferi, false middle type staphylococcus, corruption
Raw staphylococcus, Staphylococcus simiae, imitate staphylococcus, staphylococcus warneri, stenotrophomonas maltophilia,
Streptococcus agalactiae, streptococcus dysgalactiae, streptococcus dysgalactiae are like horse subspecies, streptococcus equi, streptococcus pneumoniae, micrococcus scarlatinae, breast
Room streptococcus, Streptococcus zooepidermicus, Taylorella asinigenitalis, horse reproductive tract Taylor
Salmonella, treponema carateum, spirochaeta cuniculi, Treponema hyodisenteriae, Tyreponema pallidum,
Treponema suis, Veillonella atypica, different Veillonella, veillonella parvula, rat Veillonella, vibrio cholera,
Vibrio parahaemolytious, Vibrio vulnificans, Yersinia enterocolitica, Yersinia pestis and pseudoconcretion yersinia genus
Bacterium.It is highly preferred that the list of the bacterial isolates choosing freely following species composition of described step a): Acinetobacter bauamnnii, Aerugo are false
Zymomonas mobilis and staphylococcus aureus.It is highly preferred that described bacterial isolates is the bacterial isolates of Acinetobacter bauamnnii, it is named
Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor Novoa Santos in Bu Dapei
It is deposited in Spain's Type Tissue Collection with strain number 8588 on April 14th, 2014 under this treaty.It is highly preferred that
Described bacterial isolates is the bacterial isolates of Pseudomonas aeruginosa, its named Pseudomonas aeruginosa DeltaPA4662, and by
Fundaci ó n Profesor Novoa Santos under budapest treaty with strain number 8589 on April 14th, 2014
It is deposited in Spain's Type Tissue Collection.It is highly preferred that described bacterial isolates is the Bacteria of staphylococcus aureus
Strain, its named 132deltamurI/deltadat, and reached in cloth by Fundaci ó n Profesor Novoa Santos
It is deposited in Spain's Type Tissue Collection with strain number 8587 on June 11st, 2014 under this treaty of wearing.
Additionally, the another embodiment of a first aspect of the present invention relates to one is suitable as vaccine candidate object for preparation
The method (hereafter " for preparing the method for living toxic-reduced bacteria bacterial strain of the present invention ") of living toxic-reduced bacteria bacterial strain, described method bag
Include following steps:
A. provide and can express glutamate racemase and possible D-amino acid transaminase and comprise peptidoglycan cell wall
Bacterial isolates;And
B. it is no longer able to expressive function glutamate racemase and/or functional D-amino acid transaminase with bacterial isolates
Mode inactivates the gene of encoding glutamate racemase or multiple gene, and if it is required, inactivation codase D-aminoacid turns ammonia
The gene of enzyme or multiple gene
It is auxotrophic that therefore the inactivation of wherein said gene causes described bacterial isolates to become D-Glu.
In a preferred embodiment of " method for preparing the living toxic-reduced bacteria bacterial strain of the present invention ", step a's) is thin
Bacteria strain is Gram-positive or gram negative bacteria.
In another preferred embodiment of " method for preparing the living toxic-reduced bacteria bacterial strain of the present invention ", step a's)
Bacterial isolates has the sole mode that glutamate racemase synthesizes as D-Glu, and the most described method includes that inactivation encodes this
Plant the gene of enzyme, the i.e. gene of encoding glutamate racemase.
In a further preferred embodiment of " method for preparing the living toxic-reduced bacteria bacterial strain of the present invention ", step a)
Bacterial isolates selected from the bacterial species list that consists of: Acinetobacter bauamnnii, Acinetobacter baylyi, acetic acid
Calcium acinetobacter calcoaceticus, acinetobacter haemolyticus, Acinetobacter junii, acinetobacter lwoffii, Acinetobacter nosocomialis,
Acinetobacter pittii, radioprotective acinetobacter calcoaceticus, actinobacillus lignieresii, actinobacillus suis, Aeromonas caviae, addicted to water
Aeromonas, Aeromonas veronii subsp.Sobria, actinobacillus actinomycetem comitans, Arcobacter butzleri,
Arcobacter nitrofigilis, bacillus amyloliquefaciens, anthrax bacillus, Bacillus bataviensis, solution fibrous bud
Spore bacillus, bacillus cereus, Bacillus clausii, Bacillus licheniformis, bacillus megaterium, Bacillus pumilus, withered
Grass bacillus cereus, bacillus thuringiensis, bacteroides fragilis, Bordetella avium, bordetella branchiseptica, pertussis are won
Moral spy Salmonella, Bordetella petrii, Bacillus abortus, brucella melitensis, Brucella suis, primary gram of Hall of Bulbus Allii Cepae
Moral bacterium, glanders burkholderia, Burkholderia multivorans, Burkholderia Pseudomallei,
Burkholderiathailandensis, simple and clear Campylobacter spp, campylobacter fetus fetus subspecies, campylobacter fetus sexually transmitted disease (STD) subspecies, fibre
Thin Campylobacter spp, Campylobacter hominis, campylobacter jejuni, rectum Campylobacter spp, Showa Campylobacter spp, Uppsala bending
Bacillus, citrobacter freundii, kirschner citric acid bacillus, Clostridium asparagiforme, bacillus botulinus, butyric acid shuttle
Shape bacillus, clostridium difficile, clostridium perfringens, Clostridium
Saccharobutylicum, Clostridium tetani, diphtheria corynebacterium, Corynebacterium pseudotuberculosis, clostridium perfringen,
Enterobacter cloacae, enterococcus faecalis, enterococcus faecalis, erysipelothrix ruhsiopathiae, escherichia coli, actinomyces pseudonecrophorus, fusobacterium nucleatum, adjoin
Particle chain bacterium, severe foster particle chain bacterium, horse reproductive tract haemophilus, hemophilus influenza, haemophilus parainfluenzae, the secondary bloodthirsty bar of chicken
Bacterium, haemophilus parasuis, pleuropneumonia haemophilus, Haemophilus somnus, helicobacter pylori, Klebsiella oxytoca, pneumonia
Klebsiella, Oak Ridge legionella, legionella pneumophilia, leptospira biflexa, leptospira illini, kidney hook end spiral
Body, Listeria monoeytogenes, capitate lysine bacillus cereus, spherical lysine bacillus cereus, cattle catarrhalis, rub root
Salmonella, mycobacterium abscessus, mycobacterium africanum, Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae, tuberculosis branch bar
Bacterium, gonococcus, Neisseria meningitidis, pasteurella multocida, Plesiomonas shigelloides, propionibacterium acnes,
Proteus hanseri, proteus mirabilis, Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella enteritidis intestinal subspecies,
Salmonella enteritidis, salmonella paratyphi, salmonella typhi, Serratia plymuthica, shigella boydii, Shigella dysenteriae, not
Family name's shigella dysenteriae), A Erlaite staphylococcus, staphylococcus aureus, head staphylococcus, Staphylococcus caprae, meat Fructus Vitis viniferae ball
Bacterium, staphylococcus epidermidis, Staphylococcus equorum, staphylococcus haemolyticus, staphylococcus hominis, road Deng's staphylococcus, Pasteur's Fructus Vitis viniferae ball
Bacterium, Staphylococcus pettenkoferi, false middle type staphylococcus, staphylococcus saprophyticus, Staphylococcus
Simiae, imitate staphylococcus, staphylococcus warneri, stenotrophomonas maltophilia, streptococcus agalactiae, streptococcus dysgalactiae, stop breast
Streptococcus is like horse subspecies, streptococcus equi, streptococcus pneumoniae, micrococcus scarlatinae, streptococcus uberis, Streptococcus
Zooepidermicus, Taylorella asinigenitalis, horse reproductive tract Taylor Salmonella, treponema carateum, rabbit
Treponema, Treponema hyodisenteriae, Tyreponema pallidum, Treponema suis, Veillonella atypica,
Different Veillonella, veillonella parvula, rat Veillonella, vibrio cholera, vibrio parahaemolytious, Vibrio vulnificans,
Yersinia enterocolitica, Yersinia pestis and yersinia pseudotuberculosis.
In even more preferably in embodiment of " for preparing the method for living toxic-reduced bacteria bacterial strain of the present invention ", institute
State the list of the bacterial isolates choosing freely following species composition of step a): Acinetobacter bauamnnii, Pseudomonas aeruginosa and golden yellow
Staphylococcus.
In a further preferred embodiment of " method for preparing the living toxic-reduced bacteria bacterial strain of the present invention ", described step
Rapid bacterial isolates a) is the bacterial isolates of the Acinetobacter bauamnnii of named ATCC 17978, and described method includes inactivation
Locus A1S_0380 and A1S_3398 (see embodiment 2).
In a further preferred embodiment of " method for preparing the living toxic-reduced bacteria bacterial strain of the present invention ", described step
Rapid bacterial isolates a) is the bacterial isolates of the Pseudomonas aeruginosa of named PAO1, and described method includes inactivating PA4662
Gene (see embodiment 15).
In a further preferred embodiment of " method for preparing the living toxic-reduced bacteria bacterial strain of the present invention ", described step
Rapid bacterial isolates a) is the bacterial isolates of the staphylococcus aureus of named 132, and described method includes inactivating murI
With dat gene (see embodiment 22).
In addition state and as in text, because they the most unexpected attenuations but immunogenic spy
Levy, as the bacterial isolates of present invention definition is highly suitable as the basis of attenuated vaccine alive.
About the bacterial isolates of the present invention mentioned in previous paragraph as the purposes of vaccine, the invention further relates to,
I.e. as a second aspect of the present invention, toxicity-reducing medicament compositions of living, attenuated vaccine compositions of particularly living, it comprises as fixed herein
The Auxotrophic mutant bacterial isolates of justice.
These compositionss are particularly suitable for for the wild-type form protection animal infecting sudden change auxotrophic bacterium
And people.This kind of animal can be selected from placental (including people), marsupial and monotreme.This kind of medicine group
Compound, particularly vaccine combination comprise the living toxic-reduced bacteria as defined herein of immunogenicity effective dose.Except immunogenicity
The above-mentioned living toxic-reduced bacteria of effective dose, the pharmaceutical composition of the present invention, particularly vaccine combination also comprise pharmaceutically acceptable
Carrier.Such carrier can be simple as water, but it can also such as comprise the culture fluid wherein cultivating antibacterial.Another
Suitably carrier is the solution of such as physiological salt concentration.
Useful dosage to be administered can be according to the age of immunity inoculation, body weight and animal, mode of administration and immunity inoculation
For pathogen type and change.
Described pharmaceutical composition, the most described vaccine can comprise any dosage thin that be enough to cause immunne response
Bacterium.103-1010The dosage of individual antibacterial is the most most suitable dosage.
It is optionally possible to one or more compounds with adjuvanticity are added to described pharmaceutical composition, especially
It it is vaccine.Adjuvant is immune nonspecific stimulation thing.They strengthen host's immunne response to vaccine.Known in the art
The example of adjuvant be Freund completely and Freund's incomplete adjuvant, vitamin E, non-ionic block polymer, muramyldipeptide, ISCOM
(immunostimulating complex see for example European patent EP 109942), saponin, mineral oil, vegetable oil and carbopol.The suitableeest
The adjuvant closing mucosal administration is such as HLT (LT) or cholera toxin (CT).Other suitable adjuvants are examples
Such as aluminium hydroxide, aluminum phosphate or aluminium oxide, oil-Emulsion (such asOr Saponin or Viamin E
solubilisate。
Other examples of the pharmaceutically acceptable carrier or diluent that can be used for the present invention include stabilizer such as SPGA, carbon water
Compound (such as sorbitol, mannitol, starch, sucrose, glucose, glucosan), albumen such as albumin or casein, comprise egg
White material such as Ox blood serum or skimmed milk and buffer (such as phosphate buffer).Particularly when this kind of stabilizer is added
During to vaccine, vaccine is especially suitable for lyophilization.Therefore, in a preferred form, vaccine is freeze-dried.
In order to be administered to animal or people, the pharmaceutical composition of the present invention, particularly vaccine can be with intranasal, Intradermal, subcutaneous, mouths
Take, given by aerosol or intramuscular.In order to be applied to poultry, wing lattice and eye drop are administered and are especially suitable for.The medicine of the present invention
Thing, pharmaceutical composition or vaccine may be used for asymptomatic patient and shown those patients of disease symptoms.
Therefore, a second aspect of the present invention relates to a kind of pharmaceutical composition, preferably a kind of vaccine, and it comprises D-Glu battalion
Support deficient mutants bacterial isolates alive and pharmaceutically acceptable carrier or diluent and the adjuvant being optionally present, wherein said
Pharmaceutical composition is suitable for animal and/or the people infection for the wild-type form of the sudden change auxotrophic bacterium of described compositions
Preventative (before infection) and/or therapeutic treatment (after infection or infect caused by disease clinical manifestation it
Afterwards).
In a preferred embodiment of a second aspect of the present invention, described pharmaceutical composition is vaccine, and described epidemic disease
Seedling optionally comprises adjuvant.
In the another embodiment or its any preferred embodiment of a second aspect of the present invention, described pharmaceutically acceptable
Carrier or diluent selected from the list that consists of: water, culture fluid, the solution of physiological salt concentration and/or stabilizer are such as
SPGA, carbohydrate (such as sorbitol, mannitol, starch, sucrose, glucose, glucosan), albumen such as albumin or cheese egg
In vain, protein-contg material such as Ox blood serum or skimmed milk and buffer (such as phosphate buffer) are wrapped.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described adjuvant selects
List from consisting of: Freund completely and Freund's incomplete adjuvant, vitamin E, non-ionic block polymer, muramyldipeptide,
ISCOM (immunostimulating complex), saponin, mineral oil, vegetable oil, Carbopol, escherichia coli (E.coli) heat-labile toxin
(LT) or cholera toxin (CT), aluminium hydroxide, aluminum phosphate or aluminium oxide, oil-Emulsion (such asOrSaponin and the oil-Emulsion of Viamin E solubilisate).
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described medicine group
It is 10 that compound comprises dosage3-1010The D-Glu Auxotrophic mutant of individual antibacterial bacterial isolates alive.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described medicine group
Compound is freeze-dried.
In another preferred embodiment or its any preferred embodiment of a second aspect of the present invention, described Bacteria
Select good strains in the field for seed from the bacterial species list consisted of: Acinetobacter bauamnnii, Acinetobacter baylyi, calcium acetate not lever
Bacterium, acinetobacter haemolyticus, Acinetobacter junii, acinetobacter lwoffii, Acinetobacter nosocomialis,
Acinetobacter pittii, radioprotective acinetobacter calcoaceticus, actinobacillus lignieresii, actinobacillus suis, Aeromonas caviae, addicted to water
Aeromonas, Aeromonas veronii subsp.sobria, actinobacillus actinomycetem comitans, Arcobacter butzleri,
Arcobacter nitrofigilis, bacillus amyloliquefaciens, anthrax bacillus, Bacillus bataviensis, solution fibrous bud
Spore bacillus, bacillus cereus, Bacillus clausii, Bacillus licheniformis, bacillus megaterium, Bacillus pumilus, withered
Grass bacillus cereus, bacillus thuringiensis, bacteroides fragilis, Bordetella avium, bordetella branchiseptica, pertussis are won
Moral spy Salmonella, Bordetella petrii, Bacillus abortus, brucella melitensis, Brucella suis, primary gram of Hall of Bulbus Allii Cepae
Moral bacterium, glanders burkholderia, Burkholderia multivorans, Burkholderia Pseudomallei,
Burkholderia thailandensis, simple and clear Campylobacter spp, campylobacter fetus fetus subspecies, campylobacter fetus sexually transmitted disease (STD) subspecies,
Very thin Campylobacter spp, Campylobacter hominis, campylobacter jejuni, rectum Campylobacter spp, Showa Campylobacter spp, Uppsala are curved
Curved bar bacterium, citrobacter freundii, kirschner citric acid bacillus, Clostridium asparagiforme, bacillus botulinus, butyric acid
Clostruidium, clostridium difficile, clostridium perfringens, Clostridium
Saccharobutylicum, Clostridium tetani, diphtheria corynebacterium, Corynebacterium pseudotuberculosis, clostridium perfringen,
Enterobacter cloacae, enterococcus faecalis, enterococcus faecalis, erysipelothrix ruhsiopathiae, escherichia coli, actinomyces pseudonecrophorus, fusobacterium nucleatum, adjoin
Particle chain bacterium, severe foster particle chain bacterium, horse reproductive tract haemophilus, hemophilus influenza, haemophilus parainfluenzae, the secondary bloodthirsty bar of chicken
Bacterium, haemophilus parasuis, pleuropneumonia haemophilus, Haemophilus somnus, helicobacter pylori, Klebsiella oxytoca, pneumonia
Klebsiella, Oak Ridge legionella, legionella pneumophilia, leptospira biflexa, leptospira illini, kidney hook end spiral
Body, Listeria monoeytogenes, capitate lysine bacillus cereus, spherical lysine bacillus cereus, cattle catarrhalis, rub root
Salmonella, mycobacterium abscessus, mycobacterium africanum, Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae, tuberculosis branch bar
Bacterium, gonococcus, Neisseria meningitidis, pasteurella multocida, Plesiomonas shigelloides, propionibacterium acnes,
Proteus hanseri, proteus mirabilis, Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella enteritidis intestinal subspecies,
Salmonella enteritidis, salmonella paratyphi, salmonella typhi, Serratia plymuthica, shigella boydii, Shigella dysenteriae, not
Family name's shigella dysenteriae, A Erlaite staphylococcus, staphylococcus aureus, head staphylococcus, Staphylococcus caprae, meat Fructus Vitis viniferae ball
Bacterium, staphylococcus epidermidis, Staphylococcus equorum, staphylococcus haemolyticus, staphylococcus hominis, road Deng's staphylococcus, Pasteur's Fructus Vitis viniferae ball
Bacterium, Staphylococcus pettenkoferi, false middle type staphylococcus, staphylococcus saprophyticus, Staphylococcus
Simiae, imitate staphylococcus, staphylococcus warneri, stenotrophomonas maltophilia, streptococcus agalactiae, streptococcus dysgalactiae, stop breast
Streptococcus is like horse subspecies, streptococcus equi, streptococcus pneumoniae, micrococcus scarlatinae, streptococcus uberis, Streptococcus
Zooepidermicus, Taylorella asinigenitalis, horse reproductive tract Taylor Salmonella, treponema carateum, rabbit
Treponema, Treponema hyodisenteriae, Tyreponema pallidum, Treponema suis, Veillonella atypica,
Different Veillonella, veillonella parvula, rat Veillonella, vibrio cholera, vibrio parahaemolytious, Vibrio vulnificans,
Yersinia enterocolitica, Yersinia pestis and yersinia pseudotuberculosis.
It is highly preferred that the list of the bacterial isolates choosing freely following species composition of described step a): Acinetobacter bauamnnii, copper
Green pseudomonas and staphylococcus aureus.It is highly preferred that described bacterial isolates is the bacterial isolates of Acinetobacter bauamnnii, its life
Entitled Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor Novoa Santos in cloth
It is deposited in Spain's Type Tissue Collection with strain number 8588 on April 14th, 2014 under Da Peisi treaty.More excellent
Selection of land, described bacterial isolates is the bacterial isolates of Pseudomonas aeruginosa, its named Pseudomonas aeruginosa DeltaPA4662, and
And by Fundaci ó n Profesor Novoa Santos under budapest treaty with strain number 8589 in April, 2014
Within 14th, it is deposited in Spain's Type Tissue Collection.It is highly preferred that described bacterial isolates is the thin of staphylococcus aureus
Bacteria strain, its named 132deltamurI/deltadat, and by Fundaci ó n Profesor Novoa Santos in
It is deposited in Spain's Type Tissue Collection with strain number 8587 on June 11st, 2014 under budapest treaty.
A third aspect of the present invention relates to living toxic-reduced bacteria bacterial strain, and it passes through the method for a first aspect of the present invention or passes through
Any preferred embodiment of a first aspect of the present invention obtains and maybe can obtain.
One different embodiments of a third aspect of the present invention relates to the attenuation D-Glu nutrition of living being suitable as vaccine
Deficiency bacterial isolates, it is characterised in that in described bacterial strain, the gene of encoding glutamate racemase and codase D-amino
The gene (if present) inactivation of acid transaminase.
Described inactivation can be to insert, lack, replace or combinations thereof, as long as cause can not expressive function for described inactivation
Property glutamate racemase and functional D-amino acid transaminase albumen.Functional glutamate racemase and/or functional D-amino
Acid apotransminase is interpreted as the albumen with the regulation feature of wild-type protein.It is therefore contemplated that defect therefore can not participating in
Glutamate racemase and/or the functional D-amino acid transaminase albumen of D-Glu synthesis are non-functional proteins.
In a preferred embodiment of a third aspect of the present invention, described bacterial isolates is Gram-positive or gram
Negative bacteria.
In another preferred embodiment of a third aspect of the present invention, described bacterial isolates has glutamate racemase and makees
For the sole mode of D-Glu synthesis, the most described antibacterial is characterised by that inactivation encodes the gene of this enzyme, i.e. encodes paddy
The gene of propylhomoserin racemase.
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is selected from consisting of
Bacterial species list: Acinetobacter bauamnnii, Acinetobacter baylyi, acinetobacter calcoaceticus, acinetobacter haemolyticus, fine jade
Family name's acinetobacter calcoaceticus, acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacter pittii, anti-spoke
Penetrate acinetobacter calcoaceticus, actinobacillus lignieresii, actinobacillus suis, Aeromonas caviae, Aeromonas hydrophila, Aeromonas
Veronii subsp.Sobria, actinobacillus actinomycetem comitans, Arcobacter butzleri, Arcobacter
Nitrofigilis, bacillus amyloliquefaciens, anthrax bacillus, Bacillus bataviensis, solve fiber bacillus cereus, waxy
Bacillus cereus, Bacillus clausii, Bacillus licheniformis, bacillus megaterium, Bacillus pumilus, bacillus subtilis,
Bacillus thuringiensis, bacteroides fragilis, Bordetella avium, bordetella branchiseptica, Bordetella pertussis,
Bordetella petrii, Bacillus abortus, brucella melitensis, Brucella suis, Burkholderia cepacia, glanders
Burkholderia, Burkholderia multivorans, Burkholderia Pseudomallei, Burkholderia
Thailandensis, simple and clear Campylobacter spp, campylobacter fetus fetus subspecies, campylobacter fetus sexually transmitted disease (STD) subspecies, very thin Campylobacter spp,
Campylobacter hominis, campylobacter jejuni, rectum Campylobacter spp, Showa Campylobacter spp, Uppsala Campylobacter, Freund
Citric acid bacillus, kirschner citric acid bacillus, Clostridium asparagiforme, bacillus botulinus, butyric acid shuttle shape brood cell's bar
Bacterium, clostridium difficile, clostridium perfringens, Clostridium saccharobutylicum, tetanus
Clostruidium, diphtheria corynebacterium, Corynebacterium pseudotuberculosis, clostridium perfringen, enterobacter cloacae, enterococcus faecalis, dung intestinal
Coccus, erysipelothrix ruhsiopathiae, escherichia coli, actinomyces pseudonecrophorus, fusobacterium nucleatum, adjacent particles chain bacterium, severe foster particle chain bacterium, horse
Reproductive tract haemophilus, hemophilus influenza, haemophilus parainfluenzae, haemophilus paragallinarum, haemophilus parasuis, pleuropneumonia
Haemophilus, Haemophilus somnus, helicobacter pylori, Klebsiella oxytoca, Klebsiella Pneumoniae, Oak Ridge legionella, addicted to
Lung legionella, leptospira biflexa, leptospira illini, leptospria interrogans, Listeria monoeytogenes, hammer
Shape lysine bacillus cereus, spherical lysine bacillus cereus, cattle catarrhalis, morganella morganii, mycobacterium abscessus, Africa branch
Bacillus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae, mycobacterium tuberculosis, gonococcus, meningitis Neisser
Salmonella, pasteurella multocida, Plesiomonas shigelloides, propionibacterium acnes, Proteus hanseri, proteus mirabilis,
Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella enteritidis intestinal subspecies, Salmonella enteritidis, salmonella paratyphi, wound
Cold Salmonella, Serratia plymuthica, shigella boydii, Shigella dysenteriae, shigella flexneri), A Erlaite staphylococcus, golden yellow
Color staphylococcus, head staphylococcus, Staphylococcus caprae, Staphylococcus carnosus, staphylococcus epidermidis, Staphylococcus equorum, molten
Blood staphylococcus, staphylococcus hominis, road Deng's staphylococcus, Staphylococcus pasteuri, Staphylococcus pettenkoferi,
False middle type staphylococcus, staphylococcus saprophyticus, Staphylococcus simiae, imitation staphylococcus, walsh Fructus Vitis viniferae ball
Bacterium, stenotrophomonas maltophilia, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus dysgalactiae are like horse subspecies, streptococcus equi, pneumonia chain
Coccus, micrococcus scarlatinae, streptococcus uberis, Streptococcus zooepidermicus, Taylorella
Asinigenitalis, horse reproductive tract Taylor Salmonella, treponema carateum, spirochaeta cuniculi, Treponema
Hyodisenteriae, Tyreponema pallidum, Treponema suis, Veillonella atypica, different Veillonella, little Wei are flourish
Coccus, rat Veillonella, vibrio cholera, vibrio parahaemolytious, Vibrio vulnificans, Yersinia enterocolitica,
Yersinia pestis and yersinia pseudotuberculosis.
At even more preferably in embodiment of a third aspect of the present invention, described bacterial isolates choosing freely following thing
The list of kind composition: Acinetobacter bauamnnii, Pseudomonas aeruginosa and staphylococcus aureus.
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is named ATCC
The bacterial isolates of the Acinetobacter bauamnnii of 17978, it is characterised in that inactivated gene seat A1S_0380 and A1S_3398 is (see embodiment
2)。
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is the copper of named PAO1
The bacterial isolates of green pseudomonas, it is characterised in that inactivation PA4662 gene (see embodiment 15).
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is the gold of named 132
The bacterial isolates of Staphylococcus aureus, it is characterised in that inactivation murI and dat gene (see embodiment 22).
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is Acinetobacter bauamnnii
Bacterial isolates, its named Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor
Novoa Santos is deposited in Spain typical case with strain number 8588 on April 14th, 2014 under budapest treaty and cultivates
Thing preservation center.
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is Pseudomonas aeruginosa
Bacterial isolates, its named Pseudomonas aeruginosa DeltaPA4662, and by Fundaci ó n Profesor Novoa
Santos is deposited in Spain's American Type Culture Collection with strain number 8589 on April 14th, 2014 under budapest treaty
Center.
In a further preferred embodiment of a third aspect of the present invention, described bacterial isolates is staphylococcus aureus
Bacterial isolates, its named 132deltamurI/deltadat, and by Fundaci ó n Profesor Novoa
Santos is deposited in Spain's American Type Culture Collection with strain number 8587 on June 11st, 2014 under budapest treaty
Center.
A fourth aspect of the present invention relates to the bacterial isolates as defined in a third aspect of the present invention, as medicine, especially
It is used as vaccine.
A fifth aspect of the present invention relates to pharmaceutical composition or a third aspect of the present invention of a second aspect of the present invention
D-Glu Auxotrophic mutant live bacterial isolates, for for infect described compositions sudden change auxotroph thin
The animal of the wild-type form of bacterium and/or the prophylactic treatment (before infection) of people and/or therapeutic treatment are (after infection or sense
After the clinical manifestation of the disease caused by dye) method.
A sixth aspect of the present invention relates to antibody or it is selected from Fab, F (ab') 2, the sheet of Fv, scFv, di-scFv and sdAB
Section, it is capable of identify that D-Glu Auxotrophic mutant is lived bacterial isolates, wherein said antibody or its fragment be suitable for for
The animal of wild-type form and/or the prophylactic treatment of people that infect the sudden change auxotrophic bacterium of described compositions (infect
Before) and/or therapeutic treatment (after infection or infect caused by disease clinical manifestation after).
A seventh aspect of the present invention relates to antibody or it is selected from Fab, F (ab') 2, the sheet of Fv, scFv, di-scFv and sdAB
Section, it obtains after bacterial isolates immunising mammals of living with D-Glu Auxotrophic mutant and maybe can obtain, wherein
Described antibody or its fragment are suitable for the animal of the wild-type form for the sudden change auxotrophic bacterium infecting described compositions
And/or the prophylactic treatment (before infection) of people and/or the therapeutic treatment (clinic of the disease after infection or caused by infecting
After performance).
In a preferred embodiment of a seventh aspect of the present invention, the mammal for immunity is selected from Placenta Hominis suckling
Animal (including people), marsupial and monotreme.
A eighth aspect of the present invention relates to a kind of pharmaceutical composition, preferably a kind of vaccine, its comprise the present invention the 6th or
The antibody of either side or its fragment and pharmaceutically acceptable carrier or diluent and the adjuvant being optionally present in 7th aspect,
Wherein said pharmaceutical composition is suitable for the dynamic of the wild-type form for the sudden change auxotrophic bacterium infecting described compositions
Thing and/or preventative (before the infection) of people and/or therapeutic treatment (the clinical table of the disease after infection or caused by infection
After now).In a preferred embodiment of a ninth aspect of the present invention, described pharmaceutical composition is vaccine, wherein said epidemic disease
Seedling optionally comprises adjuvant.
A ninth aspect of the present invention relates to antibody or its fragment of the 6th or the 7th aspect of the present invention, is used for treating, special
It is not for passive immunity.
A tenth aspect of the present invention relates to the pharmaceutical composition of a ninth aspect of the present invention or the 6th of the present invention or
The antibody of seven aspects or its fragment, for the wild-type form for the Auxotrophic mutant antibacterial infecting described compositions
Animal and/or the prophylactic treatment (before infection) of people and/or the therapeutic treatment (disease after infection or caused by infecting
Clinical manifestation after) method.
A eleventh aspect of the present invention relates to the second of the present invention or the pharmaceutical composition of eighth aspect or the present invention
The D-Glu Auxotrophic mutant of the third aspect live bacterial isolates or the present invention the 6th or the 7th aspect in arbitrary
The antibody of aspect or its fragment, for the wild-type form for the Auxotrophic mutant antibacterial infecting described compositions
The prophylactic treatment (before infection) of animal and/or people and/or therapeutic treatment be (disease after infection or caused by infecting
After clinical manifestation) method, and wherein said compositions, bacterial isolates or antibody or its fragment intranasal, Intradermal, skin
Under, oral, be administered by aerosol, intramuscular, wing lattice (wing web) and eye drop.
In this sense, to Acinetobacter bauamnnii (A.baumanii), Pseudomonas aeruginosa (P.aeuginosa) and gold
3 kinds of D-Glu auxotrophic strains of Staphylococcus aureus kind observe the change of IgG serum levels, and these levels depend on
Whether vaccine is by intraperitoneal, intramuscular, subcutaneous or nasal administration.Similarly, the vaccine timetable (agent of administration of administration
Amount composition and frequency) terminal level's (see embodiment 34) of produced antibody can be affected.Therefore, can for every kind of pathogen
Can be it needs to be determined that optimal timetable and the immunisation route of optimum antibody titre.For this purpose, in order to close UNICOM
Crossing humoral response and Vaccine effectiveness that the route of administration in addition to intraperitoneal (the most conventional use) obtains, author assessment passes through flesh
The use of the Δ PA4662 vaccine strains that (optimization approach being administered in people) is administered in meat, because this causes and intra-peritoneal route
Similar high-level IgG (see embodiment 18).In this respect, such as front Pseudomonas aeruginosa PAO1 Wild-Type Challenge mice.Attack
After hitting, author observes that in the mice group of non-immunity inoculation 100% is dead, and the mice of all immunity inoculations shows
100% survival (see Figure 50).This result show utilize be administered intramuscular route immunity inoculation at least with intra-peritoneal route one
Sample is effective.
Furthermore, it is noted that the medicine of the present invention, pharmaceutical composition or epidemic disease compositions Seedling may be used for asymptomatic patient and
Those patients of disease symptoms are shown.
Additionally, the author of the present invention is surprisingly found that the reagent of antibody or its fragment comprising the present invention by use
Box or device, described test kit or device allow the most qualitative and/or that quantitative analysis is individual biological sample, particularly suspect
Suffers from the bacterial species in the individual blood plasma of bacterial origin disease.
Therefore, another aspect of the present invention, i.e. a twelveth aspect of the present invention relate to antibody or its sheet comprising the present invention
The test kit of section or device are used for qualitative and/or quantitatively determine the biological sample from mammal, particularly suspect and suffer from carefully
Bacterial species in the blood plasma of the mammal of bacterial diseases.
One preferred embodiment of a twelveth aspect of the present invention relates to a kind of for being come by immunoassay detection antibacterial
The test kit that source is infected, it comprises:
I () obtains according to the 6th or the 7th aspect of the present invention or obtainable referred to as being capable of identify that of " capture antibody " is drawn
Acting the first antibody of the bacterial species infected, wherein said first antibody is preferably attached to solid support;
(ii) being referred to as second traget antibody of " detection antibody ", it identifies the district in addition to the region of first antibody identification
Territory, wherein said second antibody comprises labelling, and described labelling can be fluorescence, shiner or enzyme;
(iii) show the reagent of the affinity to second antibody, described reagent be coupled to combine to the first member;With
And
(iv) combine to the second member, it is coupled to fluorescence, shiner or enzyme, and wherein said combination is to being selected from: half
Antigen and antibody;Antigen and antibody;Biotin and avidin;Biotin and Succ-PEG-DSPE;Biotin class
Like thing and avidin;Biotin analog and Succ-PEG-DSPE;Sugar and agglutinin;Enzyme and cofactor;Nucleic acid
Or nucleic acid analog and complementary nucleic acid or nucleic acid analog.
In the context of the present invention, first antibody be referred to as " capture antibody ", its represent this antibody be used for from all carefully
The bacterial species that described antibody specificity combines given for change by the sample of fungus kind.Actually to can serve as capturing the antibody class of antibody
Type does not limit, as long as it obtains according to the seventh aspect of the invention.The antibody being suitable as capturing antibody includes but not limited to
Below: " complete antibody ", " Fab " fragment, " F (ab') 2 " fragment, " Fv " fragment, Single-Chain Fv Fragment of Murine or " scFv ", " double antibody "
And " bi-specific antibody ".
Routine techniques known in the art can be utilized to be modified further by all these antibody fragments, such as, by making
By amino acid whose disappearance, insert, replace or add and/or recombinate and/or any other modification known in the art (is such as translated
Afterwards and chemical modification, change such as glycosylation and phosphorylation).
The antibody being suitable as capturing antibody includes polyclone and monoclonal antibody.In order to prepare polyclonal antibody, permissible
By various host immunes, including goat, rabbit, rat, mice, camel, one-humped camel, yamma, people, bird etc..According to host species,
Various adjuvants can be used to increase immunne response.
In order to prepare monoclonal antibody, it is possible to use routine techniques.Such as, monoclonal antibody can utilize Kohler et
First the hybridoma method described in al, Nature, 256:495 (1975) utilizes Ausubel, FM et al. (Current
Protocols in Molecular Biology,John Wiley&Sons Inc.;Rings edition, 2003) 11.4
The process described in detail to Unit 11.11 obtains.Or, monoclonal antibody can from utilizing McCafferty et al,
The phage antibody library that technology described in Nature 348:552-554 (1990) produces is separated by recombinant DNA.
Clacksoii et al, Nature, 352:624-628 (1991) and Marks et al, J.Mol.Biol, 222:581-597
(1991) respectively describe and utilize phage library mice and people's antibody.Publication subsequently describes by chain reorganization preparation height
Affinity people's antibody (in nM scope) (Marks et al, Bio/Technology, 10:779-783 (1992)), and group
Close and infect with In vivo recombination as strategy (Waterhouse et al, the Nucl Acids building the biggest phage library
Res,21:2265-2266(1993)).Therefore, these technology are for separating the conventional hybridization of the monoclonal antibody of monoclonal antibody
The feasible selection of tumor technology.
A thirteenth aspect of the present invention relates to the test kit of a twelveth aspect of the present invention or the purposes of device, for qualitative
And/or quantitatively determine the biological sample from mammal, particularly suspect in the blood plasma of the mammal suffering from bacterial disease
Bacterial species or bacterial isolates.
Finally, a fourteenth aspect of the present invention relates to a kind of side cultivating the auxotrophic bacterial isolates of D-Glu
Method, described method includes the D-Glu utilizing variable concentrations.
In a preferred embodiment of a fourteenth aspect of the present invention, the concentration range of D-Glu is 0.00001-
120mM。
In a further preferred embodiment of this respect of the present invention, the concentration range of described D-Glu is 0.01-
50mM。
Even further preferably, the concentration range of described D-Glu is 10-20mM.
Finally, another aspect of the present invention relates to the D-Glu Auxotrophic mutant bacterial isolates that kills or it kills
The immune fragment of dead antibacterial and the purposes of these antibacterials or its immune fragment are for purposes of the present invention.
The purpose of following example only illustrates the present invention, is not to limit the present invention.
Embodiment
Embodiment 1.The nucleotide of the gene of coding Acinetobacter bauamnnii ATCC 17978 glutamate racemase and aminoacid The analysis of sequence and qualification
The author of the present invention utilizes Protein Knowledgebase (UniProtKB) database search tools to Bao Man
In acinetobacter calcoaceticus ATCC 17978 genome annotation, the gene of encoding glutamate racemase scans for.Identify corresponding to albumen
Two aminoacid sequences (locus A1S_0380 and locus A1S_3398) of MurI.By Clustal Omega comparison work
These sequences are compared by tool mutually, and other glutamate racemases present in other bacterial species with gene order-checking
Protein sequence compares.
As the result analyzed in the past, identify encoding glutamate racemization in Acinetobacter bauamnnii ATCC 17978 genome
The gene of enzyme.Consequently found that two candidate genes of possible glutamate racemase: annotate the coding into A1S_0380 and be referred to as
Gene A 1S_3398 of the gene of 288 amino acid proteins of murI and coding also referred to as 266 amino acid proteins of murI.
Fig. 4 illustrates the comparison of the aminoacid sequence of different glutamate racemase albumen, including Acinetobacter bauamnnii ATCC
The MurI (locus A1S_0380) and MurI (locus A1S_3398) and coli strain K12 of 17978 and Aerugo are false
The glutamate racemase gene of aeromonas strain PAO1.The locus A1S_0380 of Acinetobacter bauamnnii ATCC 17978 encodes
MurI albumen to enjoy mutually 29.8% at amino acid sequence level similar with the MurI albumen of coding in locus A1S_3398
Property, and they MurI albumen with escherichia coli (bacterial strain K12) are respectively provided with 33.2% and 23.2% similarity, and and copper
The MurI albumen of green pseudomonas (bacterial strain PAO1) is respectively provided with 35.8% and 37.5% similarity.
Embodiment 2.There is no structure and the sign of the different Acinetobacter bauamnnii mutant strains of glutamate racemase gene
Suicide vector pMo130 is utilized to carry out dual group of homology to build different mutant strains.First, lack independently
A1S_0380murI and A1S_3398murI gene, it is thus achieved that be called Δ 0380 and the mutant strain of Δ 3398.Expanded by PCR
Increase the fragment of about 1.000bp in the upstream and downstream region corresponding to each gene to build these mutants.By two fragments
It is cloned into carrier pMo130, utilizes the recombiant plasmid of gained to remove A1S_0380murI and A1S_ by dual group of homology
3398murI gene.
A1S_0380murI base is obtained by PCR amplification oligonucleotide UP0380 (NotI) F and UP0380 (BamHI) R
Because of fragment upstream, by the PCR primer of NotI and BamHI Restriction Enzyme digestion gained.By PCR amplification oligonucleotide
DOWN0380 (BamHI) F and DOWN0380 (SphI) R obtains A1S_0380murI downstream of gene fragment, limits with BamHI and SphI
The PCR primer of property enzymic digestion gained processed.The locus A1S_0380murI upstream region of gene of digestion and segments downstream are connected into
Front NotI and SphI linearizing carrier pMo130, it is thus achieved that be referred to as the recombiant plasmid of pMo130UP/DOWN0380.
Recombiant plasmid pMo130UP/DOWN3398 is obtained in the way of identical with former case.In this case use
Oligonucleotide is that UP3398 (NotI) F_II and UP3398 (BamHI) R_II is for fragment upstream amplification and oligonucleotide
DOWN3398 (BamHI) F and DOWN3398 (SphI) R is for the segments downstream amplification of locus A1S_3398murI gene.
By electroporation, plasmid pMo130UP/DOWN0380 and pMo130UP/DOWN3398 is introduced e. coli tg1.
The different TG1 bacterial strains converted with every kind of foregoing plasmid are cultivated in the LB with kanamycin (50 μ g/mL) at 37 DEG C
18h.Spray the bacterium colony of gained with catechol (0.45M), and only select the xylE reporter gene of expression plasmid pMo130
Yellow color colonies.Oligonucleotide UP0380 (NotI) F and DOWN0380 (SphI) R is for introducing yellow kanamycin by electroporation
Upstream and downstream fragment amplification while the plasmid pMo130UP/DOWN0380 of resistance TG1 bacterium colony.In turn, oligonucleotide
UP3398 (NotI) F_II and DOWN3398 (SphI) R is upstream and downstream sheet while plasmid pMo130UP/DOWN3398
Section amplification.
As described above, plasmid pMo130UP/DOWN0380 and pMo130UP/ of the TG1 bacterial strain of conversion will be derived from
DOWN3398 is introduced separately into Acinetobacter bauamnnii ATCC 17978 by electroporation.At the LB with kanamycin (50 μ g/mL)
Medium agar selects cointegrate bacterium colony and sprays to select yellow color colonies (XylE with catechol (0.45M)+).For with
After the independent parsing of cointegrate bacterium colony, these are inoculated in the LB culture medium of 1mL, and grow under 37 DEG C and stirring
Few 4 hours.By culture serial dilution and be inoculated in (the sacB comprised in plasmid pMo130 in the LB agar comprising 15% sucrose
Gene gives the sensitivity to sucrose).Spray the bacterium colony of gained with catechol, and (resolved by pcr analysis white colony
Cointegrate) to confirm respectively by plasmid pMo130UP/DOWN0380 and pMo130UP/DOWN3398 and allele A1S_
Δ 0380 and Δ 3398 that the equipotential exchange of 0380 and A1S_3398 produces lack.Oligonucleotide EXTFW0380 and EXTRV0380
And INTFW0380 with INTRV0380 is used for confirming that Δ 0380 suddenlys change.Oligonucleotide EXTFW3398 Yu EXTRV3398 and
INTFW3398 with INTRV3398 is used for confirming that Δ 3398 suddenlys change.
According to the previously described scheme constructs double-mutant (Δ 0380/ Δ 3398) for building single mutant.Pass through
Plasmid pMo130UP/DOWN3398 is introduced mutant Δ 0380, and the parsing cointegrate bacterium colony such as carried out in the past by electroporation,
Except making bacterium colony with or without growing on the LB agar of 10mM D-Glu to identify possible Acinetobacter bauamnnii ATCC
17978 double-mutant Δ 0380/ Δs 3398, because the bacterium colony with double sudden change needs this compound with growth.
With following oligonucleotide to by locus Δ 0380, mutant Δ 3398 in PCR confirmation mutant Δ 0380
Locus Δ 0380 and the existence of Δ 3398 in locus Δ 3398 and double-mutant, and corresponding wild type gene seat is not
Exist: EXTFW0380/EXTRV0380, INTFW0380/INTRV0380, EXTFW3398/EXTRV3398, INTFW3398/
INTRV3398, UP0380 (NotI) F/DOWN0380 (SphI) R, UP3398 (NotI) F_II/DOWN3398 (SphI) R, often
Depend on the circumstances in the case of Zhong.
Fig. 5 illustrates that the result of the PCR carried out is to confirm to exist in 3 Acinetobacter bauamnnii ATCC17978 bacterial strains built
Different sudden changes.Confirmed every kind of new mutation bacterial strain (Δ 0380, the Δ 3398 built by PCR with oligonucleotide mentioned above
With Δ 0380/ Δ 3398) in wild type gene seat A1S_0380murI or A1S_3398murI or respective mutation variants Δ
0380 and the existence of Δ 3398.
Different mutant strain cultivations under the presence or absence of D-Glu show at Acinetobacter bauamnnii ATCC
The term single gene losing encoding glutamate racemase in 1797 has no effect on the growth of this antibacterial.But, double-mutant Δ
0380/ Δ 3398 is auxotrophic, needs external source in the medium to add D-Glu to allow growth.Fig. 6 illustrates use
Double-mutant Δ 0380/ Δ 3398 genotype selects the method for bacterium colony.This bacterial strain is only supplementing the LB agar of D-Glu
Middle growth.This feature is used for selecting to have the cointegrate bacterium colony of single Δ 0380 sudden change, and it need not D-Glu next life
Long.
Generally speaking, the result of gained confirms two wild type gene seats, i.e. A1S_0380murI and A1S_ of research
In 3398murI, the existence of any one is enough for growth in the LB agar do not add D-Glu, and two bases
While cause, disappearance makes this bacterial strain can not grow in LB agar.Confirm Acinetobacter bauamnnii ATCC's 17978 in turn
A1S_0380murI and A1S_3398murI gene is to be responsible for the unique gene that D-Glu generates in this bacterial strain.
Embodiment 3.D-Glu is to the growth in double-mutant (Δ 0380/ Δ 3398) liquid medium within and viability Impact
In order to determine growth and Viability curves, by Acinetobacter bauamnnii wild-type strain ATCC 17978 and double mutant bacteria
18h is cultivated in strain at 37 DEG C in the LB supplementing 10mM D-Glu.Bacterial cultures is centrifuged, precipitation LB is washed
Twice and adjust to OD600nm=0.1.Subsequently 100 μ L are inoculated in or without in the 100mL liquid LB of 10mM D-Glu, and
And these cultures are cultivated under 37 DEG C and stirring, within every 60 minutes, continue 7 hours and finally gather sample after 24 hours
To determine the optical density of culture medium.Abreast, within every 2 hours, gather sample and be up to 6 hours, and last collection the most after 24 hours is another
One sample is to determine the CFU (colony-forming units) in the LB agar with 10mM D-Glu.All cultivations are the most triplicate
Carry out.
The growth curve making wild-type strain ATCC 1797 and mutant strain Δ 0380/ Δ 3398 is not deposited to evaluate
D-Glu effect as the function of time and these bacterial strains the existence of this compound and in the absence of existence
Power.Observe the bacterial growth (figure being completely absent double-mutant Δ 0380/ Δ 3398 in the culture medium do not have D-Glu
7A).But, the growth in the presence of D-Glu of this bacterial strain is similar to wild-type strain growth under the same conditions.When
Analyze double-mutant Δ 0380/ Δ 3398 cell survival 6 in the absence of D-Glu little constantly, it was observed that unlike wild
Type bacterial strain, due to the restriction of this compound, double-mutant viability is remarkably decreased (1log10) in first 4 hours cultivate
(Fig. 7 B).
Embodiment 4.By Acinetobacter bauamnnii wild-type strain ATCC 17978 and double mutant bacteria of electron microscopy The morphological analysis of strain (Δ 0380/ Δ 3398)
In order to shoot microphotograph by scanning electron microscopy (SEM) (SEM), by Acinetobacter bauamnnii wild-type strain
ATCC 17978 and double mutant strain cultivate 18h at 37 DEG C in the LB supplementing 10mM D-Glu.By bacterial cultures
Centrifugal, precipitation 0.1%NaCl is washed twice and is suspended in LB.The last culture of 50 μ L is inoculated in having of 5mL
0;0.1;In the LB of the D-Glu of 1.25 and 10mM concentration.Culture is cultivated 2 hours under 37 DEG C and stirring, is centrifuged subsequently
And wash twice with PBS.Then 4% paraformaldehyde in precipitation 0.1M PBS pH 7.4 is fixed 30min.After Gu Ding,
Sample is washed twice with PBS again, and by each sample at the ethanol (50%, 70%, 90% and 100%) increasing series
In each dehydration 10 minutes.Then by sample CO2(Bal-Tec CPD 030) is dried to critical point, fixing in aluminum holder,
And it is coated (Bal-Tec SCD 004 plated film instrument (sputter coater)) with one layer of gold.Utilize Jeol JSM-6400 transmission
Ultramicroscope carries out observing and shooting photo.
In order to shoot microphotograph by transmission electron microscope art (TEM), double-mutant Δ 0380/ Δ 3398 is being mended
Fill and the LB agar of 10mM D-Glu has been cultivated at 37 DEG C 18h.Carry out being passaged to MH agar subsequently, and by it 37
Incubation 18h at DEG C.After incubation, 2-3 bacterium colony is dissolved in PBS, by centrifugal for suspension and by the precipitation of gained first
Wash with dimethyl arsenic acid buffer liquid, cell is at room temperature fixed on is prepared in 0.2M sodium cacodylate buffer liquid the most immediately
In cold 2.5% glutaraldehyde in pH 7.4 4 hours.Then precipitation dimethyl arsenic acid buffer liquid is washed, be dehydrated in acetone
And be embedded in SPURR (Spurr ' s epoxy resin embedding medium).Obtain the ultrathin section (70nm) of these samples, and will
They are used for observing under JEOL JEM 1010 (80kV) transmission electron microscope(TEM) with uranyl acetate and lead citrate dyeing.
Under microscopic level, along with the external source supply of D-Glu reduces, observe aobvious in bacterial strain Δ 0380/ Δ 3398
The morphology write and structure change.Using of two kinds of bacterial strains that Fig. 8 cultivates in the presence of being shown in the D-Glu of variable concentrations scans
The microphotograph of microscope photographing.Therefore, in fig. 8 a, it can be seen that double-mutant Δ 0380/ in the absence of D-Glu
How Δ 3398 can not divide.The bacterial cell seen in having the figure of 0mM D-Glu makes people expect in the past to have this
The inoculum of compound growth.In the presence of 0.1mM D-Glu, bacterial cell shows some growths, although it is very
Slowly and the strangest, having an irregular schizotype, wherein they form cell thread with atypia binary fission
Aggregation.It was additionally observed that (disappearance of cell wall stays only by the membrane-enclosed bacterial protoplast of cell, protoplasm in protoplast group
Body).In the presence of 1.25mM D-Glu, (more Seedling height speed is anti-to see bigger cell density about aforementioned condition
Reflect), but there is many protoplasts.Some cell aggregations with atypia binary fission continue visible, although and number
Measure less, but part cell has the outward appearance that the wild type state to them is similar now.Finally, 10mM is being supplemented
In the culture medium of D-Glu, in level of density and morphocytology level, it is same that all cells is respectively provided with the wild type with them
It it is the similar performance of thing.Do not observe atypical division.
Additionally, as in Fig. 8 B it can be seen that, the author of the present invention is under different proportion, at double-mutant Δ 0380/ Δ
3398 and wild-type strain the bacterial preparation with 0.1mM D-Glu in have taken several microphotograpies.This latter bacterium
Strain has typical gram-negative coccobacillus morphology and the cell division pattern of rule, it was observed that typical binary fission, height
Cell density.In the case of double-mutant, it was observed that aforementioned morphological and atypical cell division, there is low cell subsequently close
Degree.The existence of protoplast is it will be evident that because they are generally visible near the cellular forms of structural integrity, as the former
Derivative or " ghost " occurs.In these complex, the outward appearance of cell wall is different with wild-type strain, because cell surface
It is coarse and irregular.The great variety of division level is also apparent from, since it is observed that a large amount of by 3 or more unit
The cytofilament of composition.
As used electronic scanner microscope art, carry out transmission electron microscope art research, and they displays are worked as holding and do not deposited
When D-Glu, because glutamate racemase protein function inactivation, the cell wall of double-mutant Δ 0380/ Δ 3398 experiences gradually
The property entered is destroyed, and cell cracks, and bacterial death occurs subsequently.Fig. 9 illustrates the different phase of cell wall degeneration, and scope is from having change
Cell to several places showing adventitia of semirigid structure losing them of conformation rupture and shift, cracking and be intracellular
The cell of the extrusion of tolerant (particularly hereditary material).It was additionally observed that and still keep inner membrance (to have the inoculum of D-Glu growth
Remainder) and there is the cell disperseing hereditary material therein, because glutamate racemase is can to adjust at DNA gyrase
The enzyme played a role in joint.Finally, the mechanism of cytoclasis is as follows: the disappearance of cell wall stays only by the membrane-enclosed antibacterial of cell
Protoplasm (protoplast), this makes this bacterial strain become the organism that the tension variation to culture medium is especially sensitive.The most clear
The phenomenon removed occurs, and this occurs when protoplast bursts and leave the cytoplasma membrane residue of referred to as " ghost ", also producing film
With Aggregation of Liposomes body.
Embodiment 5.Acinetobacter bauamnnii wild type ATCC 17978 and double mutant strain is determined in actute infection model Δ 0380/ Δ 3398 fatal dose (LD) in BALB/c mouse
The author of the present invention is by intraperitoneal administration Acinetobacter bauamnnii wild-type strain ATCC 17978 and double mutant bacteria
Strain Δ 0380/ Δ 3398 inoculum in saline produces systemic infection in BALB/c mouse.In order to prepare inoculum, will
Antibacterial is cultivated until reaching in LB (wild-type strain) and LB+10mM D-Glu (double-mutant) under 37 DEG C and vibration
OD600nm=0.7 (this inoculum is referred to as 1X).Culture is centrifuged, and bacterial precipitation LB is washed twice.Once wash
Wash, according to former OD600nmWith 0.9%NaCl, bacterial suspension is adjusted to various dose (0.1X;1X;2X;2.5X;3X;
4X;6X;8X and 10X) and intraperitoneal inoculation (100 μ L) in female BAl BIc/c mice, (1X is interpreted as having OD600nm=0.7
Bacterial inoculum;0.1X is interpreted as the 10-times of bacterial inoculum 1X diluted;2X is interpreted as concentrating the bacterial inoculum 1X of 2-times;
The rest may be inferred).Mice 7 days is monitored to determine the survival of various dose after infection.According to observing survival in both cases
Determine fatal dose (LD), LD100It is interpreted as the minimal lethal dose of 100% mice.
Figure 10 A illustrates when with survival in various degree in mice when increasing the wild-type strain infecting mouse of dosage.Close
Gradually reducing of mouse survival is observed, it is possible to determine LD in 2X dosage100, and LD (2.5X)100It is interpreted as mouse survival
Reduce to minimum dose necessary to 0%.When the dosage being administered is more than 3X.Observe the more rapid minimizing of survival.
Figure 10 B illustrates survival in various degree in the mice infected with double-mutant Δ 0380/ Δ 3398 increasing dosage.
In the case of this bacterial strain, LD100For the bacterial inoculum of 6X, very high dose, show that this bacterial strain toxicity is less.And open country
As in the case of raw type bacterial strain, when the antibacterial dosage being administered is more than LD100Time, it was observed that the more rapid minimizing of mouse survival.
Embodiment 6.Determine that BALB/c mouse has Acinetobacter bauamnnii wild-type strain ATCC 17978 and sudden change The bacterial load in liver in the systemic infection model of bacterial strain Δ 0380, Δ 3398 and Δ 0380/ Δ 3398
As described above, by intraperitoneal administration Acinetobacter bauamnnii wild-type strain ATCC 17978 and mutant strain
Δ 0380, Δ 3398 and Δ 0380/ Δ 3398 inoculum in saline set up systemic infection in BALB/c mouse.By open country
Raw type bacterial strain and single mutant Δ 0380 and Δ 3398 are cultivated in LB, and are existed by double-mutant Δ 0380/ Δ 3398
LB+10mM D-Glu being cultivated, cultivating until reaching OD under 37 DEG C and vibration600nm=0.7 (1X).Culture is centrifuged,
And bacterial precipitation LB is washed twice.Once wash, according to former OD600nmWith 0.9%NaCl, bacterial suspension is adjusted
Whole to 2X dosage intraperitoneal inoculation (100 μ L) in male BALB/c mouse (n=8-9).12 hours by mice after infection
Put to death with penthiobarbital.Extract liver aseptic weighing, in 0.9%NaCl after homogenate, at LB agar (wild-type strain, Δ
0380 and Δ 3398) and have in the LB agar (double-mutant Δ 0380/ Δ 3398) of 10mM D-Glu and determine CFU/ gram
Liver.
In this actute infection model, observe that infection occurs, by blood fast propagation, cause mice after infection
11-30 hour dead (Figure 10 A).Based on the count of bacteria in organ, the author of the present invention can obtain the invasion and attack of different strains
Property and the measurement of replication capacity.Therefore following meansigma methods: 8.29Log is obtained10CFU/g (mice infected with wild-type strain);
6.88Log10CFU/g (mice infected with bacterial strain Δ 0380);8.06Log10CFU/g (mice infected with bacterial strain Δ 3398)
And 1.59Log10CFU/g (mice infected with bacterial strain Δ 0380/ Δ 3398) (Figure 11).
Based on these values, compared with wild-type strain, observe in the counting of bacterial strain Δ 0380 and Δ 0380/ Δ 3398
Observe significant difference (P < 0.0001, Mann-Whitney U inspection).When the bacterium analyzing double mutant strain Δ 0380/ Δ 3398
Fall to observing when counting the most drastically and significantly reduce, in average bacteriological load, wherein obtaining the minimizing of almost 7log value, and
And do not recover from the antibacterial of 44.4% animal organ.
Embodiment 7.Determine that to have Bao Man in the BALB/c mouse with double-mutant Δ 0380/ Δ 3398 preimmunization motionless Bacterial load in liver, spleen and lung in the systemic infection model of bacillus wild-type strain ATCC 17978
In order to evaluate bacterial strain Δ 0380/ Δ 3398 effect (level of protection) as vaccine, used 1X dosage at the 0th and 14 day
Saline in bacterial strain Δ 0380/ Δ 3398 intraperitoneal immunity (100 μ L) male BALB/c mouse (n=10).At the 0th and 14 day
It is administered 100uL saline in the same manner to one group of control mice (n=10).At the 21st day, by intraperitoneal injection with Bao Man not
The 4X inoculum infecting mouse of lever bacterium wild-type strain ATCC17978.Infect latter 12 hours and mice penthiobarbital is put to death.
As described above, by the liver of every mice, spleen and lung sterile-processed, and CFU is determined.Prepare as described above and adjust antibacterial
Inoculum.Cause with the Acinetobacter bauamnnii of fatal dose with double-mutant Δ 0380/ Δ 3398 preimmunization when observing
When the bacterial load highly significant of the Different Organs of the mice that ATCC 17978 infects reduces, it is therefore evident that use double-mutant Δ
The protective effect of 0380/ Δ 3398 immunity inoculation.It is true that compared with the bacterial load of non-immune mice, it was observed that immunity
The bacterial load of each organ of mice seriously reduces (P < 0.0001, Mann-Whitney U inspection) (Figure 12).
Embodiment 8.Quantitatively different immunization scheme is carried out with bacterial strain Δ 0380/ Δ 3398 by indirect ELISA For the IgG antibody of Acinetobacter bauamnnii ATCC 17978 in BALB/c mouse
In order to evaluate the antibody-mediated immunne response in different immunization scheme, by the 0th and 14 day intraperitoneal
The immune male BALB/c mouse of bacterial strain Δ 0380/ Δ 3398 (n=12) in the saline of injection (100 μ L) 1X dosage.The 0th
It is administered 100 μ L saline to one group of control mice (n=12) in the same manner with 14 days.After immunity the 7th day, from single dose
12 mices of vaccine (being administered at the 0th day) immunity obtain serum, and at the 21st day, from the vaccine of two dosage (
0th and 14 day) immunity residue mice (n=12) obtain serum, together with the blood by the control mice (n=12) of saline injection
Clearly.In order to obtain serum, take blood by mice Thiopental Anesthesia and by eye socket clump after puncturing.By centrifugation serum with
Hemocyte is also stored at 80 DEG C until its analysis subsequently.Prepare 1X, 0.5X, 0.1X, 0.05X and 0.01X in the same manner
The vaccine of dosage, and as carried out in the past, at the 0th and 14 day immune mouse (n=6/ dosage).At the 21st day, little from immunity
Mus and obtain serum by the control mice (0X) of saline injection.
IgG is carried out quantitative by indirect ELISA (ELISA).By 96-hole elisa plate Bao Man not lever
Bacterium wild-type strain ATCC 17978 " being coated ", Acinetobacter bauamnnii wild-type strain ATCC 17978 at 4 DEG C in 100mM carbon
Hydrochlorate-bicarbonate buffer, is fixed at the bottom of hole after incubation 18h in pH 9.6.Five are carried out by phosphate-buffered saline (PBS)
(5) secondary washing is to remove loose antibacterial.By by the lock solution (5% skimmed milk in PBS) in 200 μ L/ holes at 25 DEG C
Lower incubation 1h closes remaining site.The content of sucking-off flat board with lavation buffer solution (0.005%Tween 20 in PBS)
Wash 5 times.By flat board 100 μ L serum of serial dilution in dilution buffer (there is the DMEM culture medium of 5-10%FCS)
Incubation 1h at 25 DEG C.Five (5) secondary washings are carried out to remove the most unreacted antibody with lavation buffer solution.Add 100 μ L/ holes
Two anti-(mouse IgGs of peroxidase labelling) of 1/5000 dilution in dilution buffer.By it in the dark at 25 DEG C
Incubation 1h.Flat board lavation buffer solution is washed 5 times with the anti antibody removing unreacted labelling.At the bottom of the TMB with 100 μ L
Thing (peroxidase substrate) incubation 3min develops.Reaction is terminated with the 1MHCl/ hole of 50 μ L.Read ratio at 450 nm
Color reading.For the IgG titre in the case of determining every kind, seek end point titres, have and exceed blank absorbency reading (dilution is slow
Rush the absorbance of liquid) the maximum serum-dilution of the value of 0.1 value.
As discussed above, the blood sample of every mice will be collected from for being determined about Bao Man by elisa technique
Antibody (IgG) titre of acinetobacter calcoaceticus ATCC 17978, and therefore measure the ability of vaccine generation immunne response.About rather
The mice of epidemic disease inoculation, all detects the antibody for wild-type strain (Figure 13) of significant level in the mice of all immunity.
Compared with the mice of non-immunity inoculation, antibody produces at the 7th day and the 21st day (respectively at bacterial strain Δ 0380/ Δ 3398 once
After injection and after double injection) the most significantly higher.Additionally, at the 21st day (with bacterial strain Δ 0380/ Δ 3,398 2 companies on time
After continuous injection) antibody produce the antibody being noticeably greater than the 7th day (the single injection of bacterial strain Δ 0380/ Δ 3398) and produce.
On the other hand, determine that the effect of bacterial strain Δ 0380/ Δ 3398 of various dose is to determine stimulation mouse immune system
The necessary minimum dose of system.To this end, the 0th and 14 day with following dosage each immunity 6 mices group: 0.5X,
0.1X, 0.05X and 0.01X (the lowest by 2,10,20 and 100-times).Compared with the mice in matched group, exempting from 0.01X dosage
Observe between the mice group of epidemic disease that (P < 0.01, Mann-Whitney U examines the significant difference between the 21st day IgG antibody produces
Test) (Figure 14), although the relatively degree of high vaccine dose is little.This confirms to be enough to trigger than the dosage of low 100-times of 1X dosage little
IgG in Mus produces, it was demonstrated that this bacterial strain is as the effect of vaccine.
Embodiment 9.By the IgG antibody of the BALB/c mouse of bacterial strain Δ 0380/ Δ 3398 immunity about Acinetobacter bauamnnii The cross reactivity of ATCC 17978, Acinetobacter bauamnnii ATCC 19606 and Acinetobacter bauamnnii AbH12O-A2 measures
Carry out about Acinetobacter bauamnnii the ATCC 19606 and Acinetobacter bauamnnii AbH12O-A2 serum obtained in the past
And therefore ELISA is with the antibody-mediated immunne response in the BALB/c mouse of evaluation bacterial strain Δ 0380/ Δ 3398 immunity,
Measure vaccine and produce the ability of extensive immunne response.Must be pointed out Acinetobacter bauamnnii strains A bH12O-A2 be cause several
The Highly invasive bacterial strain separated in hospital's outburst of death, and it is characterised by its resistance mould about Multiple Classes of Antibiotics
Formula.To this end, " be coated " as described above, there is Acinetobacter bauamnnii strains A TCC 17978, Acinetobacter bauamnnii strains A TCC
19606 and the flat board of Acinetobacter bauamnnii strains A bH12O-A2.Five (5) secondary washings are carried out, subsequently with 5% in PBS with PBS
Skimmed milk is closed.The content of sucking-off flat board also washs 5 times with lavation buffer solution.By flat board at 25 DEG C with 100 μ L's
The test sera incubation of 1/5 dilution 1 hour in dilution buffer.Carry out five (5) secondary washings with lavation buffer solution, and add
The mouse IgG of the peroxidase labelling of 1/5000 dilution in 100 μ L/ holes.Incubation 1h at 25 DEG C in the dark by it.Will be flat
Plate washs 5 times, and by within 3 minutes, developing with the tmb substrate incubation of 100 μ L.Terminate anti-with the 1M HCl/ hole of 50 μ L
Should, and read the color sent at 450 nm.Determine end point titres the most in each case.
About Acinetobacter bauamnnii strains A TCC 19606 and Acinetobacter bauamnnii strains A bH12O-A2 obtain with about
Those similar results (Figure 15) that Acinetobacter bauamnnii strains A TCC 17978 is observed, it was observed that similar antibody titer,
And confirmation bacterial strain Δ 0380/ Δ 3398 immunity not only produce for etc. the antibody of gene wild-type strain, but also produce
For other bacterial strains such as strains A TCC 19606 and the IgG antibody of strains A bH12O-A2 with different resistance and virulence pattern.
Embodiment 10.By attacking for different Acinetobacter bauamnnii bacterial strains with Δ 0380/ Δ 3398 mutant immunity Hit protection BALB/c mouse
In order to evaluate mutant strain Δ 0380/ Δ 3398 effect as vaccine, at the 0th and 14 day to BALB/c mouse (n
=6-12) it is administered the Δ 0380/ Δ 3398 bacterial strain (1X* dosage in saline) of 100 μ L.The the 0th and 14 day control mice only phase
It is administered together saline.Second time injection after 21 days, with Acinetobacter bauamnnii strains A TCC 17978 (4X dosage in saline),
AbH12O-A2 (4X dosage in saline) and Ab307-0294 (0.75X) attacks mice independently, in order to set up in both cases
Lethal systemic infection (intraperitoneal injections of 100 μ L).After attack, monitoring mice 7 days is to determine with control mice (rather
Epidemic disease inoculation) compare, the survival rate of immunized mice.
*, in the case of with Ab307-0294 strain challenge, it was administered to the mice of immunity inoculation respectively at the 0th and 14 day
Δ 0380/ Δ 3398 bacterial strain of 0.1X and 1X dosage.
When with Acinetobacter bauamnnii ATCC 17978 strain infection of 4X dosage, first 24 hours periods are in non-immunity inoculation
Mice group in observe 11 death, this represent in this group mortality rate (n=12) of 92%.By contrast, all immunity
The mice (n=12) of inoculation all to attacking survival, overcomes infection, and this represents 100% survival (see Figure 16) rate in this group.Two groups
Between the difference of survival be height statistically significant (P < 0.0001, according to Mantel-Cox log-rank check).
Additionally, determine by whether the response produced with Δ 0380/ Δ 3398 bacterial strain immunity be enough to provide for other
The protection of Acinetobacter bauamnnii bacterial strain lethal infection, including high virulence and pathogenic bacterial strains.With AbH12O-A2 strain challenge
In the case of, first 19 hours periods observed 9 death in the mice group of non-immunity inoculation, and this represents the mortality rate (n of 100%
=9).On the other hand, the mouse survival (n=9 of all immunity inoculations;100% survival rate) (see Figure 17).Survive between two groups
Difference be height statistically significant (P < 0.0001, according to Mantel-Cox log-rank check).
In the case of have Capsular strains to attack with Ab307-0294, in first 24 hours, we have recorded non-immunity inoculation
Mice group in 100% mortality rate and in the past by 83% survival rate in the mice group of Δ 0380/ Δ 3398 bacterial strain immunity
(see Figure 18).This confirms that the Acinetobacter bauamnnii bacterial strain that can give for having notable virulence by mutant strain immunity inoculation is drawn
The protection of the systemic infection risen.Between two groups, the difference of survival is that (P < 0.0022, according to Mantel-Cox for highly significant
Log-rank checks).
All these results show can provide for infecting different groups by Δ 0380/ Δ 3398 bacterial strain immunity inoculation
The protective immunity of Acinetobacter bauamnnii bacterial strain.
Embodiment 11.The assessment of the environmental safety assessment-water osmolisis of Δ 0380/ Δ 3398 bacterial strain
Once it leaves the individuality of immunity inoculation, and the living toxic-reduced bacteria bacterial strain of the active component of composition vaccine candidate object should
Can not replicate in general environment and adhere to.In order to compare Acinetobacter bauamnnii ATCC17978 wild type and mutant strain Δ
0380/ Δ 3398 is the long-term ability followed the trail of in general environment, required observing by cell osmolisis forfeiture viability
Time we have evaluated these bacterial strains in the water of nonnutritive thing or any contribution of salt, 37 DEG C and stirring (180rpm)
Under the conditions of survival.Gather the daily sample 2 days of culture, and last 2 times weekly, be used for determining LB agar (wild-type bacteria
Strain) and the CFU counting that supplements in the LB agar (mutant strain) of 10mM D-Glu.All cultivations are carried out the most in triplicate.
Observe that the viability of mutant strain Δ 0380/ Δ 3398 substantially reduces because cultivate 5 days within and afterwards
Do not recover the antibacterial of survival.By contrast, its wild homologue, wild-type strain keep survival until cultivate the 29th day,
At the 40th day (see Figure 19) the most irrecoverable.Between two kinds of bacterial strains, (P=0.0061, according to for the difference highly significant of survival
Raw t inspection).
Embodiment 12.The evaluation of the stability of Δ 0380/ Δ 3398 bacterial strain Middle nutrition deficiency phenotype
In order to test auxotrophic irreversible to compound D-Glu of Acinetobacter bauamnnii Δ 0380/ Δ 3398
Property, make Δ 0380/ Δ 3398 bacterial strain in 100mL supplements the LB of 10mM D-Glu under optimum condition at 37 DEG C and stir
Mix and grow 8 days under condition (180rpm).Gather from this culture when incubation period starts and at the 1st, 2,7 and 8 days
Sample CFU in the LB agar determining LB agar and supplement 10mM D-Glu.All cultivations are entered the most in triplicate
OK.In the case of the hypothesis of Phenotype transformation, similar count of bacteria should be recovered over time in agar plate, with culture medium
This compound of middle presence or absence is unrelated.On the contrary, when culture plating is cultivated in the agar with or without D-Glu
Time on base, it is observed that the significant difference between the count of bacteria obtained.
In the starting stage (the 0th day) of incubation and at the 1st, 2,7 and 8 days, (supplement D-Glu in the first situation
Agar plate) under the count of bacteria significantly higher (see Figure 20) (P=0.0006, according to student t check) of gained.There is no D-
Recovering the bacterium colony of significantly lower quantity in the agar plate of glutamic acid is owing to being derived from supplementing culture medium during growth in carefully
The residual growth of this culture of accumulation in the Cytoplasm of bacterium cell.This species diversity shows over time, Δ 0380/ Δ 3398
Bacterial strain keeps the auxotroph of D-Glu, does not has to reverse the probability for wild type phenotype.
Embodiment 13.The internal removing of Acinetobacter bauamnnii Δ 0380/ Δ 3398 bacterial strain after intraperitoneal administration in mice
In order to evaluate Acinetobacter bauamnnii Δ 0380/ Δ 3398 safety as vaccine, from preparing independently of each other
The wild type of the 1X dosage in brine media and the BALB/c of Δ 0380/ Δ 3398 mutant strain intraperitoneal inoculation (100 μ L)
The blood of mice carries out count of bacteria.After 45 minutes be administered and after 2,4,6,12 and 18 hours, by mice euthanasia,
Directly obtain blood plating in or without the LB agar of 10mM D-Glu from heart, have D-Glu for seeking
Support deficient strain, useless in wild type.
It is observed that after Gei Yaoing between the count of bacteria with wild type and the acquisition of Δ 0380/ Δ 3398 bacterial strain in 45 minutes
Significant difference (see Figure 21).In the case of Δ 0380/ Δ 3398 mutant strain, bacterium colony was not had to recover more than 6 hours.These
Result shows the acceptable secure threshold being administered this bacterial strain as vaccine candidate object, because this living toxic-reduced bacteria is given at it
Remove from health in a few hours of medicine.
Embodiment 14.Identify the gene of encoding glutamate racemase in Pseudomonas aeruginosa PAO1
Albumen knowledge base (UniProtKB) and pseudomonas genome database is utilized to analyze pseudomonas aeruginosa strains
The genome sequence of PAO1 shows the glutamate racemase gene of single presumption: PA4662, and it encodes 265 amino acid proteins.
Fig. 4 compare and than the right this presumption from Pseudomonas aeruginosa PAO1 glutamate racemase predicted amino acid sequence with
Two kinds report the glutamate racemases from Acinetobacter bauamnnii ATCC 17978 and single report from e. coli k12
Glutamate racemase.The MurI albumen of the PA4662 coding of Pseudomonas aeruginosa PAO1 and the MurI of e. coli k12 coding
Albumen (MURI_ECOLI) has 37.5% amino acid sequence similarity, encodes with the A1S_0380 locus of Acinetobacter bauamnnii
MurI albumen (A3M1P5_ACIBT) there is 35.9% amino acid sequence similarity, and with the A1S_ of Acinetobacter bauamnnii
The MurI albumen of 3398 locus codings has 37.5% similarity.
Embodiment 15.The structure of Pseudomonas aeruginosa glutamate racemase defect mutant and sign
We use in vitro method to build the mutation allele of named Δ PA4662, and it has corresponding to PA4662
The in-frame deletion of coding region.By using this mutation allele of pEX18Gm allele exchange system to replace Aerugo
Corresponding wild-type allele in pseudomonad strain PAO1 chromosome.
Plasmid pEX18Gm is used for being exchanged in Pseudomonas aeruginosa PA4662 gene by the allele in PAO1 bacterial strain
Produce unmarked disappearance Δ PA4662.By cloning the upper of the PA4662 of the leap Pseudomonas aeruginosa PAO1 of length about 1kb
Two PCR fragment of trip and downstream area build pEX18GmUP/DOWNPA4662 plasmid.Utilize primer UPPA4662
(HindIII) F_II and UPPA4662 (NotI) R expands fragment upstream, and by PCR primer HindIII of gained and NotI
Digestion.Utilize DOWNPA4662 (NotI) F and DOWN0380 (XbaI) R to expand segments downstream, and the PCR primer of gained is used
NotI and XbaI digests.The upstream and downstream fragment of digestion is connected into HindIII and XbaI linearizing carrier pEX18Gm
To produce pEX18GmUP/DOWNPA4662.
First pass through conversion and pEX18GmUP/DOWNPA4662 plasmid is introduced escherichia coli S17-1.Briefly, executing
With the escherichia coli S17-1 cell 15 μ g/ml gentamycin that electricity is turned after electric pulse competence (electrocompetent)
It is used in overnight incubation at 37 DEG C.After incubation, utilize primer UPPA4662 (HindIII) F_II and DOWNPA4662 (XbaI) R
By the bacterium colony of pcr analysis gained to confirm the pEX18GmUP/DOWNPA4662 plasmid that expectation exists.
As noted above by electroporation, pEX18GmUP/DOWNPA4662 plasmid is introduced Pseudomonas aeruginosa PAO1 bacterium
Strain, and cell is cultivated 3 days in the LB with 30 μ g/ml gentamycins at 37 DEG C.By the cointegrate bacterium of independent separate
Fall to being inoculated in the LB meat soup that 1mL supplements 10mM D-Glu and 15% sucrose, and make it same in vibrate at 37 DEG C
Shi Shengchang is overnight.Then utilize NaCl 0.9% by culture serial dilution, and by diluent plating in comprising 15%
On the LB agar of sucrose and 10mM D-Glu.Solution is chosen from the LB agar plate comprising 15% sucrose and 10mM D-Glu
Single bacterium colony the part of the cointegrate of analysis are inoculated in or without the comparable position on the LB agar plate of 10mM D-Glu.Tool
The parsing thing (Resolvant) having Δ PA4662 genotype only grows on the LB agar have D-Glu, and has wild
The thing that resolves of type genotype is growing with or without on the LB agar of D-Glu.
In this pseudomonas aeruginosa strains, the single mutant of gained Δ PA4662 needs external source D-Glu for giving birth to
Long.Figure 22 explanation is used for distinguishing the only list with Δ PA4662 mutated-genotype of growth on the LB agar comprising D-Glu
The cointegrate solely resolved with need not the D-Glu cointegrate individually resolved with wild type genotype for growth
Patch is tested.
Utilize primer UPPA4662 (HindIII) F_II/DOWNPA4662 (XbaI) R;INTFWPA4662/
INTRVPA4662 and EXTFWPA4662/EXTRVPA4662 is confirmed in suitable wild type or newly constructed mutant by PCR
The existence (utilizing last primer combined sorting to illustrate in fig 23) of the in-frame deletion variant of PA4662.
Our result show that the pEX18GmUP/DOWNPA4662 plasmid utilizing very high homology is exchanged by allele
The single in-frame deletion at the PA4662 locus of Pseudomonas aeruginosa PAO1 bacterial strain introduced gives external source D-Glu
Stringent growth need.This finds to show that PA4662 is to instruct functional glutamate racemase raw in Pseudomonas aeruginosa PAO1
The unique gene become.
Embodiment 16.Determine that Pseudomonas aeruginosa wild type and Δ PA4662 glutamate racemase defect bacterial strain are in acute sense Fatal dose (LD) in the mouse model of dye.Antibody mediated immunity response (IgG) is evaluated by indirect ELISA.
Pseudomonas aeruginosa PAO1 wild type and the Δ of various dose it is administered to BALB/c mouse (n=4 mice/group)
PA4662, it is therefore an objective to determine these bacterial strains fatal dose during acute septicopyemia infects.
In order to prepare the inoculum of administration, make antibacterial at LB (wild-type strain) and the LB that supplements 10mM D-Glu
In the lower growth of 37 DEG C and vibration until OD in (Δ PA4662 mutant)600nm=0.7 (1X dosage).Then culture is centrifuged,
And bacterial precipitation LB is washed 2 times.After cell washing, according to former OD600nmValue, hangs antibacterial with NaCl 0.9%
Supernatant liquid adjusts to various dose (0,1X;0,4X;1X;4X;10X y 40X), and by intraperitoneal injection inoculation (100 μ L) in
In BALB/c mouse, (1X looks like for having OD600nmThe bacterial inoculum of=0.7,0.1X is the bacterial inoculum of 1:10 dilution, 2X
The bacterial inoculum concentrated for 2:1, etc.).
After infecting, monitor mice 7 days, and the bacterial infection of various dose is determined survival rate.Consider two kinds of feelings
The mouse survival observed under condition determines fatal dose (LD) titre of every kind of bacterial isolates, LD100Represent 100% susceptible mice
The minimum dose that meeting is dead.
In Figure 24 A it may be seen that with increase dosage Pseudomonas aeruginosa PAO1 wild-type strain infect animal
In survival in various degree.For this bacterial strain, LD100For=0.4X.It may be seen that with increasing dosage in Figure 24 B
Survival in various degree in the animal that Δ PA4662 mutant infects.For this bacterial strain, LD100For > 40X, mice can be caused
Die from the bacterial inoculum of the very high dose of septic shock (and being not due to antibacterial duplication).This shows that this bacterial strain has
There is virulence (the ratio wild strain LD greatly reduced100The dosage of high 100 times only reduces 50% survival of mice).
Additionally, we have rated antibody mediated immunity response (IgG) by indirect ELISA.To this end, by the group of 4 mices in order to
The immunity of one of lower dosage is once: 0.1X, 0.4X, 1X, 4X, 10X and 40X.As shown in figure 44, the Δ of the 0.1X of a vaccine dose
PA4662 mutant strain (1X=5x108CFU/mL) be enough to significantly trigger IgG and produce (P < 0.001), even if being administered it at vaccine
Also it is such during detection in latter 40th day.But, the vaccine dose equal to or more than 0.4X causes the IgG of higher level to produce.
As shown in figure 45, after being administered first vaccine dose (0.4X), the 7th day IgG level dramatically increases.But, when
When being administered the 2nd vaccine dose (0.4X), antibody produces significantly higher.
The different strains Δ being derived from 3 dosage (0.4X) for the 34th day finally, with regard to Pseudomonas aeruginosa
The mice of PA4662 bacterial strain (being administered with 28 days the 0th, 14) immunity inoculation and the mice of administration saline (natural law identical)
Serum carry out ELISA with measure Δ PA4662 vaccine produce extensive immunne response ability.Obtain with bacterial strain PA28562 and close
In those similar results that pseudomonas aeruginosa strains PAO1 observes, but about the Pseudomonas aeruginosa of remaining test
Bacterial strain (PA51430664, PA26132, PAST175, PA29475 and PA12142) also sees that high-caliber cross reactivity (figure
46).This confirmation bacterial strain Δ PA4662 immunity inoculation not only produces for the antibody waiting gene wild-type strain, but also produces
The IgG antibody reacted for multiple pseudomonas aeruginosa strains.
Embodiment 17.By the Pseudomonas aeruginosa PAO1 wild type of electron microscopy and Δ PA4662 mutant strain Morphological analysis
In order to obtain electron micrograph by transmission electron microscope art (TEM), Δ PA4662 mutant strain is first made to exist
Supplementing and grow 18h in the LB agar of 10mM D-Glu at 37 DEG C, last plating is in MH agar, LB and supplements
MgCl2(30mg/L) and CaCl2(75mg/L) on LB, and incubation 18h at 37 DEG C.After incubation, by 2-3 bacterium colony
It is dissolved in PBS, suspension is centrifuged and first the precipitation of gained is washed with dimethyl arsenic acid buffer liquid, and immediately will
Cell is at room temperature fixed in the 2.5% ice-cold glutaraldehyde being prepared in sodium cacodylate buffer liquid (0.2M, pH 7.4) 4
Hour.Then by precipitation with dimethyl arsenic acid buffer liquid wash, be dehydrated in acetone and be embedded in Spurr (Spurr ' s asphalt mixtures modified by epoxy resin
Fat embedding medium) in.The ultrathin section (70nm) of sample uranyl acetate and lead citrate dyeing are used at JEOL JEM
Observe under 1010 (80kV) transmission electron microscope.
Figure 25 illustrates the different phase of the cell wall degeneration of Δ PA4662 mutant strain: from the forfeiture of the conformation with change
The cell of their rigid structure to several places showing adventitia rupture and shift, cracking and cellular content (is particularly lost
Pass material) the cell of extrusion.The mechanism of Bacteria destroyed can be in the following order: 1) disappearance of cell wall stay only by interior carefully
The membrane-enclosed bacterial protoplast of born of the same parents (protoplast), this makes this cyton be completely exposed to the tension variation of culture medium;2)
Protoplast ruptures and stays the vestige of cytoplasma membrane to be referred to as " ghost ", and it can be assembled, and individually film and liposome can
See.
Embodiment 18.By protecting BALB/c mouse anti-Pseudomonas aeruginosa PAO1 with Δ PA4662 mutant immunity Strain challenge
In order to evaluate the Δ PA4662 bacterial strain effect as vaccine, it was administered to BALB/c mouse (n=8) at the 0th and 14 day
The Δ PA4662 bacterial strain (0.4X dosage in saline) of 100 μ L.Saline it is administered the most in the same manner the 0th and 14 day control mice.The
After biphasic injection 25 days, attack mice with two with Pseudomonas aeruginosa PAO1 wild-type strain (0.4X dosage in saline)
Lethal systemic infection (intraperitoneal injections of 100 μ L) is set up in the case of Zhong.After attack, monitoring mice 7 days with determine with
Control mice (non-immunity inoculation) is compared, the survival rate of immunized mice.
When infecting with the Pseudomonas aeruginosa PAO1 wild-type strain of 0.4X dosage, first 15 hours periods connect in non-immunity
Observing 8 death in the mice group planted, this represents mortality rate (n=8) of 100% in this group.By contrast, all immunity
The mice (n=8) of inoculation all to attacking survival, overcomes infection, and this represents 100% survival (see Figure 26 A) rate in this group.Two groups
Between the difference of survival be unusual statistically significant (P < 0.0001, according to Mantel-Cox log-rank check).
These results show can provide for the protection infecting Pseudomonas aeruginosa by Δ PA4662 bacterial strain immunity inoculation
Property immunity.
Confirm these results further below.First, Figure 26 illustrates with the Pseudomonas aeruginosa PAO1 of 0.4X dosage wild
The percentage survival (87.5% vaccine potency) of BALB/c mouse (n=8) after type bacterial strain intra-abdominal infection.By immunity inoculation
Mice is immune with Pseudomonas aeruginosa Δ PA4662 bacterial strain (0.4X dosage) at the 0th and 14 day, and uses wild type at the 25th day
Strain infection.It was administered saline at the 0th and 14 day to the mice of non-immunity inoculation, and is infecting with wild-type strain on the same day.*
The survival P < 0.0001 of immunity inoculation group compared with not immunity inoculation group.P-value, according to Mantel-Cox inspection, (log-rank examines
Test).Secondly, Figure 47 shows that the mice of immunity inoculation has copper after 10 hours that acute septicopyemia infects in liver, spleen and lung
The CFU of green pseudomonas substantially reduces.Make to utilize one of optimization approach being administered in people to evaluate Δ PA4662 bacterial strain
For the Vaccine effectiveness of vaccine, utilize intramuscular route the 0th, 14 and 28 days with the Δ PA4662 bacterial strain of 100 μ L (in saline
0.4X dosage) infect BALB/c mouse (n=8/ group).Saline it is administered the most in the same manner the 0th, 14 and 28 days control mice.?
34 days, venous collection blood from the jaw of all mices and non-euthanasia, separate serum and to carry out IgG as described above quantitative.
The mice group of immunity observes the significant difference between IgG antibody generation compared with matched group, and (P < 0.0001, according to unpaired
T checks) (Figure 49).
At the 35th day, attack mice with two with Pseudomonas aeruginosa PAO1 wild-type strain (0.4X dosage in saline)
Lethal systemic infection (intraperitoneal injections of 100 μ L) is set up in the case of Zhong.After attack, monitoring mice 7 days with determine with
Control mice (non-immunity inoculation) is compared, the survival rate of immunized mice.After attack, in the mice group of non-immunity inoculation
In observe 8 death, this represent in this group mortality rate of 100%.By contrast, the mice of all immunity inoculations is all to this
Plant and attack survival, overcome infection.This represents 100% survival rate (see Figure 50) in this group.Between immunity inoculation and control mice
The difference of survival is unusual statistically significant (P < 0.0001, according to Mantel-Cox log-rank check).This result shows
Utilize the intramuscular route immunity inoculation intra-peritoneal route being administered the most effective.
Embodiment 19.With Δ PA4662 vaccine antiserum passive immunity
Completed before bacterial exposure wherein immunity inoculation timetable be impossible in the case of, when individuality cannot close
When becoming antibody, or even after being exposed to pathogen, passive immunity is used to be probably useful.Use can be taken out from host
Those antibody that Δ PA4662 immunity inoculation is formed also are transferred to another receptor, and they can provide instant passive there
Immunity or help against infectious disease.
Whether we first determine the to use by oneself vaccine serum of mice of Δ PA4662 bacterial strain immunity can be used to exposing
Passive immunity mice before Pseudomonas aeruginosa.It is being infected with the Pseudomonas aeruginosa PAO1 wild-type strain of 0.4X dosage
Front 3.5h is administered the vaccine of a dosage or inmature serum by intraperitoneal injection (200 μ L) to BALB/c mouse (n=8), and
And after attacking, monitor the survival 3 days of mice.As shown in fig. 27 a, the mice of all administration of vaccines serum is all protected exempting from
In disease, and 50% mice accepting inmature serum dies from infection (P=0.025;(log-rank examines in Mantel-Cox inspection
Test)).
These results confirm when being administered before infecting, for the blood of the mice of Δ PA4662 bacterial strain immunity inoculation of using by oneself
Clear passive immunity can give the disease that protects against of significant level, and individually antibody be enough to provide for Pseudomonas aeruginosa
The protective immunity of the acute septicopyemia of PAO1.
Then, we determined that Δ PA4662 bacterial strain immunity of using by oneself mice vaccine serum whether can as medicine to
Give the non-immune mice of the lethal injection accepting Pseudomonas aeruginosa, in order to improve the prognosis of disease.As treatment, with
After the Pseudomonas aeruginosa PAO1 wild-type strain of 0.4X dosage infects, 2h and 4.5h is little to BALB/c by intravenous injection
Mus (n=7) gives the vaccine of two dosage or inmature serum (150-200 μ L).The survival of monitoring mice 2 days.Noticeable
It is that, when the treatment for the first time of administrable serum, all mices all show the Visual symptoms that acute septicopyemia infects.Such as Figure 27 B institute
Showing, the mice of all Pseudomonas aeruginosas accepting fatal dose all dies from infection, but, the mice ratio of administration of vaccines serum connects
Time-to-live notable longer (P=0.0112 by the mice of inmature serum;Mantel-Cox checks (log-rank inspection)).Cause
This, vaccine serum acute fatal disease develop after several hours in for mice provide life maintain and contribute to protecting at once
Protect.About with vaccine serum protection and treatment dependency, these results also show may it needs to be determined that optimum antibody titre and
Administration time table.
Embodiment 20.The assessment of the environmental safety assessment of Δ PA4662 bacterial strain-water osmolisis
Vaccine candidate object it should also be understood that as representing that once it leaves the individuality of immunity inoculation, it is impossible to multiple in general environment
System and a kind of antibacterial adhered to.In order to compare Pseudomonas aeruginosa PAO1 wild type and Δ PA4662 mutant strain in general environment
The ability of medium-term and long-term tracking, we have evaluated these to lose the time necessary to viability in observation by cell osmolisis
Bacterial strain survival in the water of nonnutritive thing or any contribution of salt, under 37 DEG C and stirring (180rpm).
Initially gather the daily sample 3 days of culture, gather sample the most 2 times a week until the 62nd day, and the most every
Two weeks at least one times, is used for determining LB agar (wild-type strain) and supplementing the LB agar (mutant bacteria of 10mM D-Glu
Strain) in CFU counting.All cultivations are carried out the most in triplicate.
See that the viability of Δ PA4662 bacterial strain substantially reduces because cultivate 17 days within and the most do not recover to deposit
The antibacterial lived.By contrast, its wild homologue, wild-type strain keep extensively can recovering after the 25th day cultivated (see
Figure 28), and survive until the 143rd day.
The evaluation of the stability of embodiment 21. Δ PA4662 bacterial strain Middle nutrition deficiency phenotype
In order to test the Pseudomonas aeruginosa Δ PA4662 auxotrophic irreversibility to compound D-Glu, make
Δ PA4662 bacterial strain in 100mL supplements the LB of 10mM D-Glu under optimum condition 37 DEG C and stirring (180rpm)
Lower growth 5 days.When incubation starts and the 3rd and 5 day gather from this culture sample for determine LB agar and
Supplement the CFU in the LB agar of 10mM D-Glu.All cultivations are carried out the most in triplicate.Hypothesis feelings at Phenotype transformation
Under condition, similar count of bacteria should be recovered over time in agar plate, with this chemical combination of presence or absence in culture medium
Thing is unrelated.On the contrary, when by culture plating in or without time on the agar culture medium of D-Glu, it is observed that obtain
Count of bacteria between significant difference.
In starting stage (the 0th day) of incubation and at the 3rd and 5 day, (supplement the fine jade of D-Glu in the first situation
Fat flat board) under the count of bacteria significantly higher (see Figure 29) (P=0.0059, according to student t check) of gained.There is no D-paddy ammonia
The bacterium colony recovering significantly lower quantity in the agar plate of acid is due to thin in antibacterial during being derived from supplementing culture medium growth
The residual growth of this culture of accumulation in the Cytoplasm of born of the same parents.This species diversity shows over time, and Δ PA4662 bacterial strain keeps
The auxotroph of D-Glu, does not has to reverse the probability for wild type phenotype.
Additionally, with wild type and Δ PA4662 bacterium after 1 hour in Figure 51 it is observed that after intraperitoneal administration
Significant difference between the count of bacteria that strain obtains.In the case of Δ PA4662 mutant strain, do not had bacterium colony extensive more than 1 hour
Multiple.These results show the acceptable secure threshold being administered this bacterial strain as vaccine candidate object, because this attenuation of living is thin
Bacterium is removed from blood in its several hours be administered.
Embodiment 22. do not have the single of the staphylococcus aureus of glutamate racemase and/or D-amino acid transaminase or
The structure of double mutant strains and sign
Temperature sensitive copy carrier pMAD is utilized to carry out dual group of homology to build mutant strain.First, obtain independently
The glutamate racemase (murI) of annotation and the unmarked disappearance of D-amino acid transaminase (dat).Each single mutant strain
It is called Δ murI and Δ dat.
In order to complete the structure of the two mutant, by PCR amplification corresponding to gene both sides upstream (left side) and under
The 1000bp fragment of trip (right side) DNA, and it is cloned into individually shuttle plasmid pMAD.The recombiant plasmid of gained is with making a return journey
Except being positioned at the chromosome murI on staphylococcus aureus 132 wild-type strain chromosome and dat gene.
Staphylococcus aureus 132 is that model organism as species " staphylococcus aureus " is used in the present invention
Produce the clinical MRSA bacterial strain of the Auxotrophic mutant of D-Glu.This clinical strains methicillin-resistant (MRSA)
(Vergara-Irigaray et al,Infection and Immunity,77(9):3978-3991(2009))。
MurIUP (MluI) F and murIUP (NotI) R primer is utilized to obtain murI gene to being combined through PCR amplification
Fragment upstream, is digested by MluI and NotI Restriction Enzyme subsequently.Utilize murIDOWN (NotI) F and murIDOWN
(BglII) R primer obtains the segments downstream of murI gene by PCR amplification, then digests with NotI and BglII enzyme.Will
The upstream and downstream fragment of the murI gene of digestion is cloned into former MluI and BglII enzyme linearizing pMAD carrier, it is thus achieved that
The construct of named pMAD_UP/DOWN_murI.
Complete same policy for building plasmid pMAD_UP/DOWN_dat.In this case, it is utilized respectively primer pair
DatUP (MluI) F/datUP (NotI) R and datDOWN (NotI) F/datDOWN (BglII) R amplification dat gene upstream and
Segments downstream.
By electroporation, pMAD_UP/DOWN_murI and pMAD_UP/DOWN_dat is introduced e. coli tg1.Supplementing
Ampicillin (100 μ g/mL) is plus selecting transformant bacterium colony on the LB agar of X-Gal (150 μ g/mL).Incubation at 37 DEG C
After 18h, carry out PCR with primer combination murIExtF/murIExtR or datExtF/datExtR respectively and check that ampicillin resists
The existence of pMAD_UP/DOWN_murI or pMAD_UP/DOWN_dat in property blue colonies.
By only for construct pMAD_UP/DOWN_murI and pMAD_UP/DOWN_dat that extract from e. coli tg1 cell
Before on the spot introducing, electroporation enters the middle clone strain staphylococcus aureus of target strain golden color staphylococcus 132
RN4220.The erythromycin (the 10 μ g/mL) TSB plus X-Gal (150 μ g/mL) is being supplemented after incubation 24-48h at 30 DEG C
The selection of the staphylococcus aureus RN4220 bacterium colony comprising recombiant plasmid is carried out on agar.
Extract each recombiant plasmid from staphylococcus aureus RN4220 and be independently introduced into golden yellow by electroporation
Color staphylococcus 132 wild-type strain.Equally, the Erythromycinresistant of the staphylococcus aureus 132 comprising each construct is made
Blue colonies supplement erythromycin (10 μ g/mL) plus X-Gal (150 μ g/mL) TSB agar at 30 DEG C grow 24-
48h。
In order to lack the chromosome murI gene of staphylococcus aureus 132 wild-type strain, pMAD_UP/ will be comprised
One colony lift of staphylococcus aureus 132 wild type of DOWN_murI is to the TSB with 10 μ g/mL erythromycin of 5mL
In, and make it grow 2h at 30 DEG C.Afterwards, by culture incubation at 43.5 DEG C (unlicensed temperature that pMAD replicates),
The Characteristics for Single Staggered recombination and integration causing the pMAD_UP/DOWN_murI upstream by murI gene or downstream area enters Bacterial stain
Body.After 6 hours, TSB culture serial dilution being coated on is supplemented erythromycin (10 μ g/mL) plus X-Gal (150 μ g/
ML) on TSA flat board, and incubation 18h at 43.5 DEG C.The colony lift of several gained is not had to 5mL the TSB of antibiotic
In, and at 30 DEG C incubation 18h to cause pMAD_UP/DOWN_murI to intersect thing from the second time of Chromosomal excision
Part.By serial dilution plating is no longer comprised pMAD_ on the TSB flat board with X-Gal (150 μ g/mL)
The selection of the white colony of UP/DOWN_murI.By the colony lift of each selection to having X-Gal (150 μ g/mL) and X-
Gal (150 μ g/mL) is plus on the TSA of erythromycin (10 μ g/mL).Utilize murIExtF/murIExtR, murIseqF/
MurI gene delection (the Δ of murIseqR and the murIF/murIR primer white colony to being checked erythromycin-sensitive by PCR
murI)。
PMAD_UP/DOWN_dat plasmid is still used to carry out from golden yellow Fructus Vitis viniferae according to the scheme in the past murI described
The chromosome deficiency dat gene of coccus 132 wild-type strain.In this case, utilize datExtF/datExtR,
The combination of datseqF/datseqR and datF/datR primer confirms to suspect the bacterium colony no longer comprising dat gene (Δ dat).
In order to produce double-mutant Δ murI/ Δ dat, by electroporation, pMAD_UP/DOWN_da plasmid is introduced sudden change
Δ murI bacterial strain, and the same approach single mutant described before carrying out.In this case, double-mutant is extensive
Need again to add to (being respectively 20 or 10mM for TSB or TSB agar) in culture medium external source D-Glu, because having double
The bacterium colony of gene delection needs this D-aminoacid to be used for growing.Following primer is utilized to confirm staphylococcus aureus by PCR
In the genome of 132 wild-type strains, murI and dat locus does not exists: murIExtF/murIExtR, murIseqF/
MurIseqR, murIF/murIR, datExtF/datExtR, datseqF/datseqR and datF/datR.
The single disappearance of different mutant strain display murI or dat gene is cultivated in or without the culture medium of D-Glu
Do not affect bacterial growth.But, double-mutant needs to there is D-Glu in the medium for growing.Figure 30 represents and is used for selecting
The bacterium colony screening selecting the white colony of the erythromycin-sensitive caused by second time crossing event of single mutant Δ murI and carry out
Method.
Figure 31 illustrates that in 3 kinds of mutant strains of staphylococcus aureus 132 wild type, the PCR of different disappearances confirm.To mesh
The result obtained till before confirms two wild type gene seat murI or/and the existence of any one is for golden yellow Fructus Vitis viniferae in dat
The coccus 132 normal growth in the TSB not having the D-Glu added is enough, and two genes of disappearance make simultaneously
This bacterial strain can have growth in the presence of D-Glu.In sum, it was demonstrated that the murI of staphylococcus aureus 132 and
Dat gene is the participation biosynthetic unique gene of D-Glu in this bacterial strain.
Therefore it is noted that the inactivation of glutamate racemase and D-amino acid transaminase causes the auxotrophy of D-Glu
Type, and about staphylococcus aureus species obtain Auxotrophic mutant method independent of selected bacterial strain.
Embodiment 23.D-glutamic acid is to the growth in double-mutant Δ murI/ Δ dat liquid medium within and viability
Impact
In order to assess staphylococcus aureus double-mutant Δ murI/ Δ compared with staphylococcus aureus 132 wild type
The growth of dat and Viability curves, by the two of exponential phase of growth kinds of bacterial strains with final 1/200 dilution inoculate into 100ml TSB and
Supplement the TSB of 20mM D-Glu, and incubation under 37 DEG C with persistent oscillation (180rpm).1,2,3,4,5 and 18h,
Determine optical density and the bacterial concentration of culture.By at OD600nmThe aliquot of lower measurement every bottle evaluates optical density, simultaneously
Bacterial concentration (CFU/mL) is calculated on TSB agar plate by being coated by continuous 10-times of diluent.The homogeneous formula of all cultivations
Three parts are carried out.
Carry out the growth curve of S. aureus wild type and double mutant strain Δ murI/ Δ dat with evaluate with
The time, the most there is not the impact of D-Glu, and this compound existence and in the absence of bacterial strain
Viability.
Double mutant strains are observed by the TSB culture medium do not have D-Glu and is completely absent growth (Figure 32 A), when
Recover the most completely when D-Glu is added to culture medium.About bacterial viability (Figure 32 B), according to display due in culture medium
The restriction of D-Glu, the viability of double-mutant substantially reduces (2Log at 18h10)。
Embodiment 24. passes through staphylococcus aureus 132 wild type and the double-mutant Δ murI/ Δ of electron microscopy
The morphological analysis of dat bacterial strain
In order to prepare electron microscopy sample, the golden yellow supplementing in the TSB of the D-Glu of 20mM will be recovered from
The liquid culture of aureus wild-type type and double-mutant Δ murI/ Δ dat bacterial strain washes twice in saline and is resuspended in
In the TSB of 1mL.Every kind of suspension of 50 μ L is transferred in the TSB supplementing the D-Glu increasing concentration of 5mL: 0,
0.1,1.5 and 20mM, and incubation 3 hours under 37 DEG C with persistent oscillation (180rpm).The bacterial cultures of acquisition is centrifuged,
And bacterial precipitation PBS is washed twice.Afterwards, by precipitation with the paraformaldehyde 4% in PBS 1M pH 7.4 in room temperature and
30 minutes are fixed under vibration.Sample PBS is washed twice extraly, and with the determining alcohol of a series of increases (50%,
70%, 90%, 100%) dehydration, each 15 minutes.Finally, sample is used at a critical point CO2It is dried (Bal-Tec CPD
030).One of each sample is placed on microscope slide and is fixed on aluminum holder and is used for gold and is coated (Bal-Tec SCD 004
Plated film instrument).In transmission electron microscope Jeol JSM-6400, observe sample and take pictures.
Microscope is observed and is made it possible to detect along with the D-Glu in culture medium reduces, double-mutant Δ murI/ Δ dat
In bacterial strain, morphology and the structure of essence change.Figure 33 is shown in the TSB culture medium of the D-Glu supplementing variable concentrations
After growth, compare the scanning electron image of wild type and double-mutant bacterial strain.
Figure 33 A is displayed without the D-Glu of outside interpolation, and double-mutant can not divide.Therefore, at the D-Glu of 0mM
Under, the initial inoculation thing that the bacterial cell of detection grows before nearly corresponding in the presence of this culture.Owing to expecting
Intracellular D-Glu and the incomplete fissional result that occurs, it is also possible to observe the cell shape of some exceptions.
When by double-mutant incubation in supplementing the culture medium of D-Glu of 0.1mM, some cell divisions occur, but due to
The amino acid whose low concentration of this D-, cell division continues atypia.Therefore, the process of cell wall synthesis and division cannot not be complete
Complete, the cell being therefore deformed and protoplast (lack Peptidoglycan but still comprise the bacterial cell of plasmalemma).?
In the presence of the D-Glu of 1.5mM, relative to being derived from the culture of this compound with low concentration, bacterial density is slightly
Micro-increase, shows the relatively Seedling height rate of double-mutant.The shape of protoplast and lengthening yet suffers from, it can be observed that have with
The typical sphere bacterial cell of the configuration that wild-type strain is identical.Finally, when culture media supplemented during D-Glu 20mM, double prominent
Comparable with shown by wild-type strain of the cell density of variant and morphology.Do not observe atypical schizotype.
On the other hand, the double-mutant of incubation and open country during Figure 33 B is shown in the cultivation of the D-Glu supplementing 0.1mM
The different scanning electron micrograph of raw type bacterial strain.Wild-type strain display gram-positive cocci characteristic spheroid shape,
High-cell density and typical schizotype.Staphylococcus aureus division is characterised by that incomplete cell wall separates, because of
This apparently the bacterial cell in grape cluster keep connecting by Peptidoglycan fragment.On the contrary, because the incomplete cell of aggravation divides
Splitting, the cell of double-mutant becomes the forming part of large-scale tight agglomerate or polymer.Culture medium lack sufficient concentrations of
D-Glu infringement Peptidoglycan biosynthesis also corrects cell wall division.It practice, cell density is seen than in wild type goods
Observe is low.Additionally, visible abundant deformed shape, including abnormal lengthening antibacterial.Under these conditions, double-mutant also table
Reveal coarse and irregular surface compared with wild-type strain.
Additionally, the progressively disintegrate of cell wall, antibacterial degeneration detected in double-mutant by transmission electron microscope art
Bacterial death subsequently.To this end, by the double mutant strain of short-term maintenance in the absence of D-Glu as described in Example 4
Obtain sample product.As seen from Figure 34, this method allows to observe the different progressively disintegrate stages until antibacterial is dead
Die.The initial configuration change observed includes that murein layer reduces, and result cell size (protoplast) increases, because peptide gathers
Sugar constitutes the mechanical resistance part of cell wall.It can also be observed that the permeability of plasma membrane and destruction, this causes cytoplasm fraction
Discharge.At these cell peripherals caved in it can be seen that a large amount of fragment, film aggregation, liposome and hereditary material.The opposing party
Face, some bacterial cells still keep complete pod membrane, therefore it is observed that typical S. aureus bacterium shape and
Size.
Embodiment 25. determines S. aureus wild type 132 and double-mutant Δ murI/ in actute infection model
Δ dat fatal dose (LD) in BALB/c mouse
In order to produce systemic infection in BALB/c mouse, in mouse peritoneal, it is administered S. aureus wild type
With double mutant strains there is 3% pig mucinous saline inoculum.
In order to prepare inoculum, make antibacterial in TSB (wild type) and TSB (double sudden changes of supplementing 20mM D-Glu
Body) in 37 DEG C and vibration (180rpm) under growth until reaching the OD of 0.7600nm.Culture is centrifuged, washs with saline serum
Twice, and be resuspended in there are 3% pig mucinous saline serum (wild type and the double mutant bacterias for intraperitoneal sepsis model
Strain) or saline serum (for the double-mutant of immune animal, following example) in~108The final concentration of CFU/mL.This
Suspension is referred to as 1X, and when being administered the volume of 250 μ L to mice corresponding to 5x107CFU/ mice.Bacterial suspension is entered
One successive step is to various dose (such as, suspension 3X is interpreted as 3 times of bacterial inoculums etc. concentrated).By BALB/c (n=3-4)
With the bacterial suspension intraperitoneal inoculation of various dose (250 μ L), and monitor metainfective 14 days.LD100It is defined as 100%
The minimal lethal dose of dead mouse.
The depositing in various degree when being administered the S. aureus wild type bacterial strain increasing dosage is shown in Figure 35 A
Live.Determine that the minimum dose survival of mice reduced to 0% is 3X.Figure 35 B illustrates when being administered the double of increase dosage
Survival in mice during mutant strain.By contrast, this figure shows to inoculate the LD than wild-type strain100High 10-multiple dose
Double-mutant causes 100% survival rate.Therefore, the fatal dose of double-mutant is more than 30X LD100>30X.This clearly confirms that gold
The double-mutant of Staphylococcus aureus is the highly attenuated bacterial strain showing the virulence potentiality lower than wild type counterparts bacterial strain.
Embodiment 26. utilizes systemic infection model to determine with the BALB/c of double-mutant Δ murI/ Δ dat preimmunization
Bacterial load in the spleen of mice and blood
In order to evaluate staphylococcus aureus double-mutant Δ murI/ Δ dat bacterial strain effectiveness (the protection water as vaccine
Flat), carry out two independent experiments.
First, at the 0th and 14 day by the BALB/c mouse (n=4-6) double-mutant Δ murI/ Δ in saline serum
Dat bacterial strain (10X dosage) intraperitoneal preimmunization (250 μ L).250 μ L salt it are administered in the same manner to one group of mice at the 0th and 14 day
Water.At the 21st day, with the lethal inoculum of staphylococcus aureus 132 wild-type strain, (5X dosage, 250 μ L had 3% pig and glue
Albumen) intra-abdominal infection mice.Mice used in 20 hours penthiobarbital euthanasia after infection.Take aseptic for the spleen of every mice
Go out and after homogenate in saline, by serial dilution plating being determined in TSB agar CFU/ gram of organ.By inciting somebody to action
50 μ L are inoculated in the TSB culture medium of 5mL from the blood sample of the aseptic taking-up of mouse heart and carry out the existence of antibacterial in evaluating blood.
Prepare as mentioned previously and adjust bacterial inoculum.
Cause with staphylococcus aureus 132 wild-type strain of fatal dose with this bacterial strain preimmunization when observing
When bacterial load in the mice spleen infected substantially reduces, it was demonstrated that by the protection effect of double-mutant Δ murI/ Δ dat immunity inoculation
Should.It practice, it is observed that compared with non-immune mice, the bacterial load of immune mouse seriously reduces (2Log10) (P=
0.0095, Mann-Whitney U-inspection, Figure 36 A).Additionally, there is not antibacterial in the blood of the mice of all immunity inoculations
(negative blood culture) supports the protective effect (Figure 36 B) of immunity inoculation further.
Confirm these results further below.First, Figure 37 shows with staphylococcus aureus 132 wild-type strain
Lethal inoculum (5X dosage, 250 μ L have 3% pig mucin) infect after 22 hours, with double-mutant Δ murI/ Δ
In the BALB/c mouse (n=8-9) of dat bacterial strain (8X dosage in this case) preimmunization, spleen (Figure 37 A) and blood (figure
Count of bacteria in 37B) is significantly lower compared with non-immune group, and (respectively P=0.0006 and P=0.0002, according to Mann-
Whitney U checks).Immunization schedule executed as described above.
Embodiment 27. is by protecting the anti-Staphylococcus aureus of BALB/c mouse with Δ murI/ Δ dat mutant immunity
Bacterium 132 strain challenge
In order to evaluate the Δ murI/ Δ dat effect as vaccine, at the 0th and 14 day to BALB/c mouse (n=10-13)
It is administered Δ murI/ Δ dat bacterial strain (10X dosage in saline) of 250 μ L.It is administered the most in the same manner the 0th and 14 day control mice
Saline.After second time injection 7 days, (glue having 3% pig with staphylococcus aureus 132 wild-type strain of fatal dose
5X dosage in the saline of albumen) attack mice to set up lethal systemic infection in both cases.After attack, monitor little
Mus to determine compared with matched group (non-immunity inoculation), the survival rate of immunized mice.
When infecting with staphylococcus aureus 132 wild-type strain of 5X dosage, in the mice group of non-immunity inoculation
Observing 9 death, this represents the mortality rate of 90% in this group (n=10).By contrast, the 8 of immunity inoculation group (n=13)
Mice all to attacking survival, overcomes infection, and this represents 61.5% survival rate (see Figure 38) in this group.Survive between two groups
Variant statistical notable (P < 0.031 checks according to Mann-Whitney U).
These results show can provide for postoperative infection golden yellow Fructus Vitis viniferae by Δ murI/ Δ dat bacterial strain immunity inoculation
The protective immunity of ball.
Embodiment 28. passes through indirect ELISA at the BALB/c carrying out double-mutant Δ murI/ Δ dat bacterial strain immunity inoculation
Quantitatively for the IgG antibody waiting gene staphylococcus aureus 132 Δ spa bacterial strain in mice
In order to evaluate the antibody-mediated immunne response to immunity inoculation, at the 0th and 14 day by intraperitoneal injection (250 μ
L) double-mutant Δ murI/ Δ dat (10X dosage) in saline carrys out immune BALB/c mouse (n=10).At the 21st day, by little
Mus Thiopental Anesthesia also punctures collection blood by rear eye socket clump.By centrifugation serum and hemocyte be stored in-
Until analyzing at 80 DEG C.
Indirect ELISA (ELISA) is utilized to carry out IgG detection.By the 96-hole whole golden yellow of elisa plate
Staphylococcus 132 Δ spa bacterial strain is coated.This bacterial strain be staphylococcus aureus 132 wild-type strain of A protein deficiency etc.
Gene bacterial strain.Therefore, in carbonate-bicarbonate buffer 100mM, (OD in pH 9.6 at 4 DEG C600The culture of=1
1/10 diluent) after incubation 18h by the bottom of whole bacteria adhension to hole.With phosphoric acid buffers saline solution (PBS) buffer
Carry out five (5) secondary washings to remove loose antibacterial.Two steps are carried out close remaining site to reduce and mice serum
Non-specific interaction.First, by flat board at room temperature by lock solution (5% skimmed milk in the PBS) temperature in 100 μ L/ holes
Educate 1h, then, with 100 μ L rabbit anteserum (1/1000) incubation 1h at 37 DEG C.With lavation buffer solution (0.005% in PBS
Tween 20) carry out 5 washing steps after, flat board (is had 5-10%FCS with 100 μ L in dilution buffer at 4 DEG C
DMEM culture medium) serial dilution Serum incubation overnight.Second day, carry out 5 washings with lavation buffer solution the most anti-to remove
The antibody answered, and add two anti-/ hole (the anti-mouse IgG peroxidase of 1/5000 dilution in dilution buffer of 100 μ L
HRP-labelling).At room temperature carry out incubation 1h.Flat board lavation buffer solution is washed 5 times.In order to carry out developing process, will
Flat board TMB (HRP-peroxidase substrate) the incubation 3min of 100 μ l.Reaction is terminated with the 1M HCl/ hole of 50 μ L.?
Colorimetric measurement is carried out under 450nm.Include that the positive (anti-Staphylococcus aureus monoclonal antibody), feminine gender (are come at all flat boards
Serum from the mice of non-immunity inoculation) and compare with reference to (dilution buffer).In order to determine the IgG titre of each serum, estimate
Meter end point titres.Titre is defined as having the last serum-dilution than the absorbance with reference to high 0.1 of comparison (dilution buffer).
Therefore, the blood sample of every mice will be collected from for being determined for staphylococcus aureus 132 by ELISA
The titre of the antibody (IgG) of Δ spa bacterial strain, measures vaccine from this and produces the ability of immunne response.With the mutant Δ of 10X dosage
MurI/ Δ dat immunity mice group observe compared with the mice in matched group IgG antibody produce between significant difference (P <
0.0001, check according to Mann-Whitney U) (Figure 39), it was demonstrated that this bacterial strain triggers the effect in IgG response in mice.
Embodiment 29.By the IgG antibody of double-mutant Δ murI/ Δ dat generation for unrelated S. aureus bacterium The cross reactivity of strain
ELISA is carried out to comment about USA300LAC, RF122, ED133 and ED98 bacterial strain with the serum shown in embodiment 28
Valency with Δ murI/ Δ dat bacterial strain immunity BALB/c mouse in for the unrelated S. aureus bacterium from separate sources
The antibody-mediated immunne response of strain, therefore measures vaccine and produces the ability of extensive immunne response.It is known that golden yellow Fructus Vitis viniferae
Coccus USA300LA is the popular MRSA bacterial strain causing abnormal affecting conditions in healthy individuals, is the U.S., Canada
Main cause with the Community Acquired Infections in Europe.On the other hand, RF122 (cattle, ST151 and CC151), ED133 are selected
(sheep, ST133 and CC133) and ED98 (poultry, ST5 and CC5) are as causing pathogenetic adaptation animal place in domestic animal
The representative bacterial strain of 3 dominant clone of main staphylococcus aureus strains.
To this end, working flat plate as described in Example 28, but in advance by the " bag independently of every kind in flat board above-mentioned bacterial strains
Quilt ".
As shown in figure 40, the antibody titer of highly significant detected for 4 kinds of unrelated staphylococcus aureus strains, card
Practical Δ murI/ Δ dat bacterial strain immunity not only produces for the antibody waiting gene 132 Δ spa bacterial strain, but also produces for phase
Close clinical strains USA300LAC and adapt to very much the antibody of 3 kinds of other bacterial strains of animal reservoir.
Environmental safety assessment-water the osmolisis of embodiment 30. Δ murI/ Δ dat bacterial strain and the assessment of resistance to drying condition
Once it leaves the individuality of immunity inoculation, and attenuated vaccine material standed for of living can not be adhered in general environment, mesh
Be to maintain minimum relevant risk.
In order to compare the long-term tracking in general environment of S. aureus wild type and Δ murI/ Δ dat bacterial strain
Ability, we have evaluated these bacterial strains in not battalion to lose the time necessary to viability in observation by cell osmolisis
Survival in the water of any contribution supporting thing or salt, under the conditions of room gentle agitation (180rpm).Gather sample every day of suspension
Product be used for determining TSB agar (wild-type strain) and supplement 10mM D-Glu TSB agar (mutant strain) in
CFU counts.All cultivations are carried out the most in triplicate.
As shown in figure 41, observe that the viability of mutant Δ murI/ Δ dat bacterial strain reduces, over time over time
Passage, 24 hours 2-Log10Reduce.Additionally, cultivated the Δ murI/ Δ dat bacterial strain antibacterial not recovering survival more than 72 hours.
Additionally, Figure 52 illustrates compared with wild-type strain, double-mutant Δ murI/ Δ dat bacterial strain is to dry resistance.For
Evaluation drought resistance, by will washing exponential phase of growth cell dilution series point sample to nitrocellulose filter (0.45 μm
Aperture) test wild type and the cell survival of double mutant strain.By moist for filter membrane (growth control) or in aseptic training
At 37 DEG C, it is dried 12 or 18 hours (drought stress conditions) in supporting ware, then they are placed in the TSB supplementing D-Glu
On flat board, and incubation 24 hours at 37 DEG C.All cultivations and dilution series are carried out the most in triplicate.
As it can be seen, compared with growth control (undried filter membrane), to wild-type bacteria after being maintained under drought condition
The difference of cell survival is not observed in strain.By contrast, the 2-Log of Δ murI/ Δ dat bacterial strain10The growth of dilution culture
Substantially reduce after being dried 12 hours.When be maintained under drying stress 18 little constantly, in the identical diluent of double-mutant
Observe and be completely absent cell survival (without growth).These results show that Δ murI/ Δ dat bacterial strain is than wild-type parent bacterium
Strain is more sensitive to being dried, and supports that it is used as the suitable secure threshold of vaccine further.
Embodiment 31.The evaluation of the stability of staphylococcus aureus Δ murI/ Δ dat bacterial strain Middle nutrition deficiency phenotype
Can not to the auxotrophic of compound D-Glu in order to test staphylococcus aureus Δ murI/ Δ dat
Inverse property, make Δ murI/ Δ dat bacterial strain in 100mL supplements the TSB of 20mM D-Glu under optimum condition at 37 DEG C and
Grow 11 days under stirring (180rpm).The sample from this culture is gathered when incubation starts and at the 3rd, 5 and 11 days
CFU in the TSB agar determining TSB agar and supplement 10mM D-Glu.All cultivations are carried out the most in triplicate.
In the case of the hypothesis of Phenotype transformation, similar count of bacteria should be recovered over time in agar plate, and in culture medium
This compound of presence or absence is unrelated.On the contrary, when by culture plating in the agar culture medium with or without D-Glu
Time upper, it is observed that the significant difference between the count of bacteria obtained.
In the starting stage (the 0th day) of incubation and at the 3rd, 5 and 11 days, (supplement D-Glu in the first situation
Agar plate) under the count of bacteria significantly higher (see Figure 42) of gained.This species diversity shows over time, Δ murI/ Δ dat
Bacterial strain keeps the auxotroph of D-Glu, does not has to reverse the probability for wild type phenotype.
The internal removing of staphylococcus aureus Δ murI/ Δ dat bacterial strain after intraperitoneal administration in embodiment 32. mice
In order to evaluate inoculation in vivo as the safety of staphylococcus aureus Δ murI/ Δ dat bacterial strain during vaccine
Property, compared with wild-type strain, determine thin in the spleen of BALB/c mouse, kidney and the blood by Δ murI/ Δ dat inoculation
Bacterium load.(high 13-times to wild-type strain and the 10X of the sub-lethal 0.7X dosage of mice (each bacterial strain of n=3/) intraperitoneal administration
Dosage) Δ murI/ Δ dat bacterial strain.The most independently of each other two kinds of cultures are prepared in and there is 3% pig glue egg
In white saline.After infection the 1st, 2 and 6 days by a mice/group penthiobarbital euthanasia to determine in organ and blood
Bacterial load.Therefore, as described above by spleen and kidney sterile-processed, and by by serial dilution plating in TSB
Agar (wild type) and determine CFU/ gram of organ in supplementing the TSB agar (double-mutant) of 10mM D-Glu.By by 50
μ l determines the bacterial load blood from the blood plating of the aseptic taking-up of heart.
In acute sepsis model, infect and quickly spread by blood along with antibacterial and occur, 24-48 hour after infection
Produce the peak of dead mouse.Therefore, the count of bacteria from organ and blood can obtain the aggressive of specific bacterial strain with multiple
The tolerance of ability processed.
It is observed that over time in Figure 43, the mice being administered with Δ murI/ Δ dat bacterial strain with use wild-type strain
The mice infected is compared, the significant difference between count of bacteria in kidney, spleen and blood.Even pre-when the peak-peak infected
When phase is between 24-48 hour, Δ murI/ Δ dat bacterial strain was not recovered bacterium colony in the 1st or 2 day after infection.Even if it is true that
Being administered the inoculum of 1:13 dilution, after these infect, the CFU of virulent wt bacterial strain recovers to reach maximum count.
As a result, " internal " of Δ murI/ Δ dat bacterial strain is removed and occurred before 24 hours, and this shows that it is as attenuation of living
The suitable level of security that vaccine is administered.
Embodiment 33. staphylococcus aureus 132 Δ murI/ Δ dat vaccine antiserum passive immunity
Then we have studied the vaccine of the mice of staphylococcus aureus 132 Δ murI/ Δ dat bacterial strain immunity of using by oneself
The effect that serum protection mice staphylococcus infects.
Before attacking with toxicity staphylococcus aureus 132 wild-type strain of 5X dosage, 3.5h makes vaccine or inmature blood
Clearly (150 μ L) enter the peritoneal cavity of BALB/c mouse (n=5), and monitor the survival 14 days of mice.
As shown in Figure 53, the mice of useful vaccine serum passive immunity all protected by for attack, and 60% connects
Infection (P=0.0429 is died from by the mice of inmature serum;Mantel-Cox checks (log-rank inspection)).
These results confirm the staphylococcus aureus antiserum that transfer produces with double-mutant Δ murI/ Δ dat bacterial strain
Give significant level mice protection, and with toxicity staphylococcus aureus 132 wild-type strain infect before 3.5 hours
Death is prevented during administration.
The different approaches that embodiment 34. vaccine development is administered
The timetable being administered and approach may decide that the potential immunogenicity of specific vaccine, are that vaccination finally becomes
The key factor of merit.
Therefore, we have rated and utilize the different approaches intraperitoneal of administration, intramuscular, the subcutaneous and antibody of intranasal initiation
Whether immunne response (IgG) can be affected by this variable.Moreover, it is contemplated that the quantity of administration time table, i.e. dosage and epidemic disease
Seedling consumption (bacterial inoculum content).
All approach all have merits and demerits, such as absorption, bioavailability and the metabolism of material.At present, major part approval
People's vaccine be parenteral, utilize intramuscular, subcutaneous and intradermal route to be administered.Although considering intramuscular vaccines up to now
Inoculating as final mode, nose approach provides easy automedication, inducing mucosal and systemic immunity.And, liquid
All can give by this way with dry powder formulations.Finally, intranasal administration may be the suitableeest after hyperinfection illness outbreak
Close barrier immunity inoculation.
Mucosa is extensively exposed to microorganism and other exterior materials, and therefore they are strong immunocompetent places.Bao Man is not
Lever bacterium, Pseudomonas aeruginosa and staphylococcus aureus are the common causes of respiratory tract infection (among others), therefore
Use intranasal immunization can be of value to determining of preventing that these microorganisms from causing to grow and disease.
When comparing 3 kinds of parenteral route of test, it has been found that in producing antibody-mediated immunne response intraperitoneal,
Difference is not had between subcutaneous and intramuscular adminstration.And, derive at intranasal administration Acinetobacter bauamnnii and Pseudomonas aeruginosa
Similar antibody titers is found after vaccine.By contrast, S. aureus vaccines causes relatively low by mucosal immunity
The IgG of degree produces (Figure 48 A-C).In general, repeat immune strengthening IgG over time to produce.
Consider vaccine dose, it is seen that the different mode of humoral response depends on microorganism.Utilize parenteral route (for copper
Green pseudomonas also has intra-nasal route) live attenuation Acinetobacter bauamnnii-and the Pseudomonas aeruginosa of two kinds of proof loads being administered-
Vaccine (respectively 1X and 0.1X;0.4X and 0.04X) the most effectively trigger antibody generation (Figure 48 A-B).Staphylococcus aureus-
In the case of vaccine (Figure 48 C), when lowest dose level (1X) is used for by intra-nasal route immune mouse, it was observed that IgG titre
Substantially reduce.But, even if using minimum antibacterial dosage (for intraperitoneal and intramuscular route 0.2X;And for subcutaneous 1X),
Parenteral route immunity inoculation is used to be still effectively.
The effect that mice is protected by the different approaches being administered to evaluate vaccine, once by the epidemic disease that Pseudomonas aeruginosa is derivative
The immunization time end of list (EOL) of Seedling, attacks with the wild type PAO1 bacterial strain (intraperitoneal administration as mentioned previously) of fatal dose (0.4X)
Mice, and as above monitor survival.The antibody titers that the 5th at vaccine produces after being administered as expected, all mices
Survival, it was demonstrated that the good correlation between IgG and protection.Therefore, the route of administration of all evaluations can be suitable for preventing this micro-
The acute septicopyemia that biology causes.
Sequence table
The glutamate racemase MurI (A3MIP5_ACIBT) of SEQ ID No 1: Acinetobacter bauamnnii ATCC 17978
MTAIQPLFTELEPMPKALADAPIGIFDSGIGGMSVAAEIAKYLPNERIVYYADTAYVPYGPRSDEEIRELTARAVDW
LYRQGCKIAVVACNTASAFSLDHLREHYGEHFPIVGLVPALKPAVLQTRSKVVAVLATPATFRGQLIKDVVEKFAVP
AGVKVMTLTSLELVPCVEAGQQMSPVCLNALREVLQPAVEQGADYLVLGCTHYPFLNEAIHHLFDNQFTLVDSGLAV
ARQTARILIKNELLCDQIRQNVARIECYVSGNNADALQPVLQNMIPQELTWTLHNLS
The glutamate racemase MurI (A3MA43_ACIBT) of SEQ ID No 2: Acinetobacter bauamnnii ATCC 17978
MNNNNNPIGMIDSGLGGLSLFKYIRQALPNEDIIYFADSKYVPYGDRESDWIVSRTTHLISNLVTHGKCKAIVIACN
TMTAVAVETIRAQINVPLIAIEPAVKPAVAMTLSKHIAVLATATTVKGKNLKSLIETYAQDIKVSLVPCIGLAEKIE
TGKAHTAEVKDYLKNILAPLVEQKVDTIILGCTHYPFVSDTIQEIVGRDIQIIEPSEAVTAQLIRQLNQYHLSSESP
NEGNHIIWTSSDPLEVADVTFSLLGTRLPVETTDF
SEQ ID No 3: colibacillary glutamate racemase MurI (MURI_ECOLI)
MATKLQDGNTPCLAATPSEPRPTVLVFDSGVGGLSVYDEIRHLLPDLHYIYAFDNVAFPYGEKSEAFIVERVVAIVT
AVQERYPLALAVVACNTASTVSLPALREKFDFPVVGVVPAIKPAARLTANGIVGLLATRGTVKRSYTHELIARFANE
CQIEMLGSAEMVELAEAKLHGEDVSLDALKRILRPWLRMKEPPDTVVLGCTHFPLLQEELLQVLPEGTRLVDSGAAI
ARRTAWLLEHEAPDAKSADANIAFCMAMTPGAEQLLPVLQRYGFETLEKLAVLG
The glutamate racemase MurI (MURI_PSEAE) of SEQ ID No 4: Pseudomonas aeruginosa
MAVESAAVGVFDSGVGGLSVLREIRARLPSESLLYVADNAHVPYGEKSAEYIRERCERIGDFLLEQGAKALVLACNT
ATAAAAAELRERYPQVPLVAMEPAVKPAAAATRNGRVGVLATTGTLKSARFAALLDRFASDVQVFTQPCPGLVERIE
AGDLYGPQTRALLERLLAPILEQGCDTLILGCTHYPFVKPLLAELIPAEMAVIDTGAAVARQLERVLSARALLASGQ
AATPRFWTSALPEEMERILPILWGSPESVGKLVV
The glutamate racemase MurI (MURI) of SEQ ID No 5: staphylococcus aureus 132
MNKPIGVIDSGVGGLTVAKEIMRQLPNETIYYLGDIGRCPYGPRPGEQVKQYTVEIARKLMEFDIKMLVIACNTATA
VALEYLQKTLSIPVIGVIEPGARTAIMTTRNQNVLVLGTEGTIKSEAYRTHIKRINPHVEVHGVACPGFVPLVEQMR
YSDPTITSIVIHQTLKRWRNSESDTVILGCTHYPLLYKPIYDYFGGKKTVISSGLETAREVSALLTFSNEHASYTEH
PDHRFFATGDPTHITNIIKEWLNLSVNVERISVND
D-amino acid transaminase Dat (DAT) of SEQ ID No 6: staphylococcus aureus 132
MEKIFLNGEFVSPSEAKVSYNDRGYVFGDGIYEYIRVYNGKLFTVTEHYERFLRSANEIGLDLNYSVEELIELSRKL
VDMNQIETGAIYIQATRGVAERNHSFPTPEVEPAIVAYTKSYDRPYDHLENGVNGVTVEDIRWLRCDIKSLNLLGNV
LAKEYAVKYNAVEAIQHRGETVTEGSSSNAYAIKDGVIYTHPINNYILNGITRIVIKKIAEDYNIPFKEETFTVDFL
KNADEVIVSSTSAEVTPVIKLDGEPVNDGKVGPITRQLQEGFEKYIESHSI
SEQ ID No 7:UP0380(NotI)F:CCCGCGGCCGCGGGGTCCTGCACCTACGATGA
SEQ ID No 8:UP0380(BamHI)R:CCCGGATCCGGGACCTCCAATACCTGAATC
SEQ ID No 9:DOWN0380(BamHI)F:CCCGGATCCGGGGCTCTGTTGTAGGCATTC
SEQ ID No 10:DOWN0380(SphI)R:CCCGCATGCGGGCATCCTTGTGATTGCATT
SEQ ID No 11:UP3398(NotI)F_II:CCCGCGGCCGCGGGTTGGTCAGGTCCTTGTTG
SEQ ID No 12:UP3398(BamHI)R_II:CCCGGATCCGGGTACAGCCGTCATGGTGTT
SEQ ID No 13:DOWN3398(BamHI)F:CCCGGATCCGGGACGCGTTTACCTGTAGAA
SEQ ID No 14:DOWN3398(SphI)R:CCCGCATGCGGGAGCGGTACAACTAATTGG
SEQ ID No 15:EXTFW0380:GCAATTAGGCACTTGAGG
SEQ ID No 16:EXTRV0380:ATACGCTCAGGTTGCATC
SEQ ID No 17:INTFW0380:AGCCTATGTTCCGTATGG
SEQ ID No 18:INTRV0380:TCAACCAGTGTGAATTGG
SEQ ID No 19:EXTFW3398:CCGATTGGAATGATTGAC
SEQ ID No 20:EXTRV3398:AGAGCATTCTGGTCGAAG
SEQ ID No 21:INTFW3398:TAGCAATAGAACCAGCGG
SEQ ID No 22:INTRV3398:TTGTGCCGTTACAGCTTC
SEQ ID No 23:UPPA4662(HindIII)F_II:
CCCAAGCTTGGGGGCAATCCGCCGTATATC
SEQ ID No 24:UPPA4662(NotI)R:
CCCGCGGCCGCGGGGGCGTTGCCCGCAGACGG
SEQ ID No 25:DOWNPA4662(NotI)F:
CCCGCGGCCGCGGGTCGTTCCTGGCAGACGTG
SEQ ID No 26:DOWNPA4662(XbaI)R:CCCTCTAGAGGGTCCGCTCTCGCAGTCCGA
SEQ ID No 27:EXTFWPA4662:GTATCGGCAAGGTGGAGT
SEQ ID No 28:EXTRVPA4662:GAATGGCTTGATCGAGTC
SEQ ID No 29:INTFWPA4662:ATCCGAATCGTTGCTCTA
SEQ ID No 30:INTRVPA4662:ACAATACGCGCTCCAGCT
SEQ ID No 31:murIUP(MluI)F:CCCACGCGTGGGCCGAAACAAAAAACAGTA
SEQ ID No 32:murIUP(NotI)R:CCCGCGGCCGCGGGATTCGGTCATCCTTACTT
SEQ ID No 33:murIDOWN(NotI)F:
CCCGCGGCCGCGGGGAGGATTTTTAATGAAAG
SEQ ID No 34:murIDOWN(BglII)R:CCCAGATCTGGGTTTCTTCCATTGAACTTC
SEQ ID No 35:murIF:TGTCGGAGGTTTGACAGTAG
SEQ ID No 36:murIR:CTAACTTCACGAGCCGTTTC
SEQ ID No 37:murIExtF:GCTTGCCCTAAAGGTATTCC
SEQ ID No 38:murIExtR:GGGCCACTCATACTTATGAC
SEQ ID No 39:murIseqF:ATGACTGAACAATCAGTGAA
SEQ ID No 40:murIseqR:TGATGGTGCCATGTAAAGTT
SEQ ID No 41:datUP(MluI)F:CCCACGCGTGAAACGTATTCATATGAT
SEQ ID No 42:datUP(NotI)R:CCCGCGGCCGCATATTATTCCTCCACGCA
SEQ ID No 43:datDOWN(NotI)F:CCCGCGGCCGCAATTCTTTCATCATATTT
SEQ ID No 44:datDOWN(BglII)R:CCCAGATCTGCGAATCTAAACTCGGTA
SEQ ID No 45:datF:TATTCAAGCAACGCGTGGTG
SEQ ID No 46:datR:AGTTGACGTGTAATTGGGCC
SEQ ID No 47:datExtF:GTCATGGGTGACGTGACAAC
SEQ ID No 48:datExtR:GCACCACCTGCTGAATCAAG
SEQ ID No 49:datseqF:GCCGGTTGTAACAGAAGATG
SEQ ID No 50:datseqR:CAATTGCCGGGTCTGCAATC
Claims (38)
1. prepare and comprise D-Glu Auxotrophic mutant and live the pharmaceutical composition (preferred vaccine) of bacterial isolates for one kind
Method, it is thin for the Auxotrophic mutant of described compositions that wherein said pharmaceutical composition is suitable for animal and/or people
The prophylactic treatment (before infection) of the infection of the wild-type form of bacteria strain and/or therapeutic treatment are (after infection or infect
After caused Disease Clinical performance), and wherein said pharmaceutical composition prepared by the method comprised the following steps:
A. D-Glu Auxotrophic mutant bacterial isolates alive is obtained;
B. bacterial isolates of being lived by described Auxotrophic mutant joins pharmaceutically acceptable carrier or diluent optionally
Add adjuvant;And
The most optionally pharmaceutical composition described in lyophilization.
2. prepare and comprise D-Glu Auxotrophic mutant and live the pharmaceutical composition (preferred vaccine) of bacterial isolates for one kind
Method, it is thin for the Auxotrophic mutant of described compositions that wherein said pharmaceutical composition is suitable for animal and/or people
Preventative (before infection) and/or the therapeutic treatment (after infection) of the infection of the wild-type form of bacteria strain, and wherein
Described pharmaceutical composition is prepared by the method comprised the following steps:
A. provide and can express glutamate racemase and possible D-amino acid transaminase and the antibacterial comprising peptidoglycan cell wall
Bacterial strain;
B. so that described bacterial isolates is no longer able to expressive function glutamate racemase and/or functional D-aminoacid turns ammonia
The mode of enzyme inactivates a gene of encoding glutamate racemase or multiple gene, and if it is required, inactivation codase D-ammonia
One gene of base acid transaminase or multiple gene, therefore the inactivation of wherein said gene causes described bacterial isolates to become D-paddy
Propylhomoserin auxotroph;And
C. bacterial isolates of being lived by described Auxotrophic mutant joins pharmaceutically acceptable carrier or diluent optionally
Add adjuvant;And
The most optionally pharmaceutical composition described in lyophilization.
3. the method preparing pharmaceutical composition of claim 1 or 2, wherein said pharmaceutical composition is vaccine, and described system
Preparation Method includes adding adjuvant.
4. the method any one of claim 1-3, the bacterial isolates of wherein said step a) is Gram-positive or gram
Negative bacteria.
5. the method for claim 2, the bacterial isolates of wherein said step a) has glutamate racemase and closes as D-Glu
Become sole mode, and the step b) of wherein said method include inactivation coding this enzyme, i.e. glutamate racemase gene.
6. the method any one of claim 1-3, the bacterial isolates of wherein said step a) is selected from the antibacterial consisted of
Species list: Acinetobacter bauamnnii (Acinetobacter baumannii), Acinetobacter baylyi, calcium acetate are not
Lever bacterium (Acinetobacter calcoaceticus), acinetobacter haemolyticus (Acinetobacter haemolyticus),
Acinetobacter junii (Acinetobacter junii), acinetobacter lwoffii (Acinetobacter lwoffii),
Acinetobacter nosocomialis, Acinetobacter pittii, radioprotective acinetobacter calcoaceticus (Acinetobacter
Radioresistens), actinobacillus lignieresii (Actinobacillus lignieresii), actinobacillus suis
(Actinobacillus suis), Aeromonas caviae (Aeromonas caviae), Aeromonas hydrophila (Aeromonas
Hydrophila), Aeromonas veronii subsp.Sobria, actinobacillus actinomycetem comitans (Aggregatibacter
Actinomycetemcomitans), Arcobacter butzleri, Arcobacter nitrofigilis, solution starch spore
Bacillus (Bacillus amyloliquefaciens), anthrax bacillus (Bacillus anthracis), Bacillus
Bataviensis, solution fiber bacillus cereus (Bacillus cellulosilyticus), bacillus cereus (Bacillus
Cereus), Bacillus clausii (Bacillus clausii), Bacillus licheniformis (Bacillus
Licheniformis), bacillus megaterium (Bacillus megaterium), Bacillus pumilus (Bacillus
Pumilus), bacillus subtilis (Bacillus subtilis), bacillus thuringiensis (Bacillus
Thuringiensis), bacteroides fragilis (Bacteroides fragilis), Bordetella avium (Bordetella
Avium), bordetella branchiseptica (Bordetella bronchiseptica), Bordetella pertussis
(Bordetella pertusis), Bordetella petrii, Bacillus abortus (Brucella abortus), sheep kind cloth
Shandong Salmonella (Brucella melitensis), Brucella suis (Brucella suis), Burkholderia cepacia
(Burkholderia cenocepacia), glanders burkholderia (Burkholderia mallei), Burkholderia
Multivorans, Burkholderia Pseudomallei (Burkholderia pseudomallei), Burkholderia
Thailandensis, simple and clear Campylobacter spp (Campylobacter concisus), campylobacter fetus fetus subspecies
(Campylobacter fetus subsp.fetus), campylobacter fetus sexually transmitted disease (STD) subspecies (Campylobacter fetus
Subsp.venerealis), very thin Campylobacter spp (Campylobacter gracilis), Campylobacter hominis, sky
Intestinal Campylobacter spp (Campylobacter jejuni), rectum Campylobacter spp (Campylobacter rectus), Showa Campylobacter spp
(Campylobacter showae), Uppsala Campylobacter (Campylobacter upsaliensis), Freund citric acid
Bacillus (Citrobacter freundii), kirschner citric acid bacillus (Citrobacter koseri), Clostridium
Asparagiforme, bacillus botulinus (Clostridium botulinum), Bacillus butyricus (Clostridium
Butyricum), clostridium difficile (Clostridium difficile), clostridium perfringens
(Clostridium perfringens), Clostridium saccharobutylicum, Clostridium tetani
(Clostridium tetani), diphtheria corynebacterium (Corynebacterium diphtheriae), Corynebacterium pseudotuberculosis
(Corynebacterium pseudotuberculosis), clostridium perfringen (Enterobacter aerogenes), cloaca
Enterobacteria (Enterobacter cloacae), enterococcus faecalis (Enterococcus faecalis), enterococcus faecalis
(Enterococcus faecium), erysipelothrix ruhsiopathiae (Erysipelothrix rhusiopathiae), escherichia coli, bad
Dead Fusobacterium (Fusobacterium necrophorum), fusobacterium nucleatum (Fusobacterium nucleatum), adjoin
Particle chain bacterium (Granulicatella adiacens), severe foster particle chain bacterium (Granulicatella elegans), horse reproduction
Road haemophilus (Haemophilus equigenitalis), hemophilus influenza (Haemophilus influenzae), pair
Hemophilus influenza (Haemophilus parainfluenzae), haemophilus paragallinarum (Haemophilus
Paragallinarum), haemophilus parasuis (Haemophilus parasuis), pleuropneumonia haemophilus
(Haemophilus pleuropneumoniae), Haemophilus somnus (Haemophilus somnus), helicobacter pylori
(Helicobacter pylori), Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella Pneumoniae
(Klebsiella pneumoniae), Oak Ridge legionella (Legionella oakridgensis), legionella pneumophilia
(Legionella pneumophila), leptospira biflexa (Leptospira biflexa), leptospira illini
(Leptospira illini), leptospria interrogans (Leptospira interrogans), monocyte hyperplasia Liszt
Bacterium (Listeria monocytogenes), capitate lysine bacillus cereus (Lysinibacillus fusiformis), spherical
Lysine bacillus cereus (Lysinibacillus sphaericus), cattle catarrhalis (Moraxella bovis), rub Gen Shi
Bacterium (Morganella morganii), mycobacterium abscessus (Mycobacterium abscessus), mycobacterium africanum
(Mycobacterium africanum), Mycobacterium avium (Mycobacterium avium), Mycobacterium bovis
(Mycobacterium bovis), Mycobacterium leprae (Mycobacterium leprae), mycobacterium tuberculosis
(Mycobacterium tuberculosis), gonococcus (Neisseria gonorrhoeae), Neisseria meningitdis
Bacterium (Neisseria meningitidis), pasteurella multocida (Pasteurella multocida), class will congratulate adjacent unit cell
Bacterium (Plesiomonas shigelloides), propionibacterium acnes (Propionibacterium acnes), Proteus
Hanseri, proteus mirabilis (Proteus mirabilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa),
Salmonella choleraesuis (Salmonella cholerasuis), Salmonella enteritidis intestinal subspecies (Salmonella enterica
Subsp.enterica), Salmonella enteritidis (Salmonella enteritidis), salmonella paratyphi
(Salmonella paratyphi), salmonella typhi (Salmonella typhi), Serratia plymuthica (Serratia
Plymuthica), shigella boydii (Shigella boydii), Shigella dysenteriae (Shigella dysenteriae), Freund
Shigella dysenteriae (Shigella flexneri), A Erlaite staphylococcus (Staphylococcus arlettae), golden yellow Portugal
Grape coccus (Staphylococcus aureus), head staphylococcus (Staphylococcus capitis), goat Fructus Vitis viniferae ball
Bacterium (Staphylococcus caprae), Staphylococcus carnosus (Staphylococcus carnosus), staphylococcus epidermidis
(Staphylococcus epidermidis), Staphylococcus equorum (Staphylococcus equorum), haemolysis Fructus Vitis viniferae ball
Bacterium (Staphylococcus haemolyticus), staphylococcus hominis (Staphylococcus hominis), road Deng's Fructus Vitis viniferae ball
Bacterium (Staphylococcus lugdunensis), Staphylococcus pasteuri (Staphylococcus pasteuri),
Staphylococcus pettenkoferi, false middle type staphylococcus (Staphylococcus
Pseudointermedius), staphylococcus saprophyticus (Staphylococcus saprophyticus), Staphylococcus
Simiae, imitation staphylococcus (Staphylococcus simulans), staphylococcus warneri (Staphylococcus
Warneri), stenotrophomonas maltophilia (Stenotrophomonas maltophilia), streptococcus agalactiae
(Streptococcus agalactiae), streptococcus dysgalactiae (Streptococcus dysgalactiae), streptococcus dysgalactiae
Like horse subspecies (Streptococcus dysgalactiae subsp.equisimilis), streptococcus equi (Streptococcus
Equi), streptococcus pneumoniae (Streptococcus pneumoniae), micrococcus scarlatinae (Streptococcus
Pyogenes), streptococcus uberis (Streptococcus uberis), Streptococcus zooepidermicus,
Taylorella asinigenitalis, horse reproductive tract Taylor Salmonella (Taylorella equigenitalis), pinta are close
Spirillum (Treponema carateum), spirochaeta cuniculi (Treponema cuniculi), Treponema
Hyodisenteriae, Tyreponema pallidum (Treponema pallidum), Treponema suis, Veillonella atypica
(Veillonella atypica), different Veillonella (Veillonella dispar), veillonella parvula (Veillonella
Parvula), rat Veillonella (Veillonella ratti), vibrio cholera (Vibrio cholerae), vibrio parahaemolytious
(Vibrio parahaemolyticus), Vibrio vulnificans, Yersinia enterocolitica (Yersinia
Enterocolitica), Yersinia pestis (Yersinia pestis) and yersinia pseudotuberculosis (Yersinia
pseudotuberculosis)。
7. the method for claim 6, the list of the bacterial isolates choosing freely following species composition of wherein said step a): Bao Man is not
Lever bacterium, Pseudomonas aeruginosa and staphylococcus aureus.
8. the method for claim 7, the bacterial isolates that bacterial isolates is Acinetobacter bauamnnii of wherein said step a), its name
For Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor Novoa Santos according to cloth
Da Peisi treaty is deposited in Spain's Type Tissue Collection with strain number 8588 on April 14th, 2014.
9. the method for claim 7, the bacterial isolates that bacterial isolates is Pseudomonas aeruginosa of wherein said step a), its name
For Pseudomonas aeruginosa DeltaPA4662, and by Fundaci ó n Profesor Novoa Santos according to Budapest bar
About it is deposited in Spain's Type Tissue Collection with strain number 8589 on April 14th, 2014.
10. the method for claim 7, the bacterial isolates that bacterial isolates is staphylococcus aureus of wherein said step a), its
Named 132deltamurI/deltadat, and by Fundaci ó n Profesor Novoa Santos according to Budapest
Treaty is deposited in Spain's Type Tissue Collection with strain number 8587 on June 11st, 2014.
11. 1 kinds of pharmaceutical compositions, preferably a kind of vaccine, its comprise D-Glu Auxotrophic mutant live bacterial isolates with
And pharmaceutically acceptable carrier or diluent and the adjuvant being optionally present, wherein said pharmaceutical composition be suitable for animal and/
Or people (infects it for the preventative of infection of the wild-type form of the Auxotrophic mutant bacterial isolates of described compositions
Before) and/or therapeutic treatment (after the clinical manifestation of the disease after infection or caused by infection).
The pharmaceutical composition of 12. claim 11, wherein said pharmaceutical composition is vaccine, and wherein said vaccine is optionally
Comprise adjuvant.
Pharmaceutical composition any one of 13. claim 11-12, wherein said pharmaceutically acceptable carrier or diluent are selected from
The list consisted of: water, culture fluid, the solution of physiological salt concentration and/or stabilizer such as SPGA, carbohydrate are (such as
Sorbitol, mannitol, starch, sucrose, glucose, glucosan), albumen such as albumin or casein, the protein-contg material of bag such as
Ox blood serum or skimmed milk and buffer (such as phosphate buffer).
Pharmaceutical composition any one of 14. claim 11-13, wherein said adjuvant is selected from the list consisted of: not
Family name completely and Freund's incomplete adjuvant, vitamin E, non-ionic block polymer, muramyldipeptide, ISCOM (immunostimulating complex),
Saponin, mineral oil, vegetable oil, Carbopol, HLT (LT) or cholera toxin (CT), aluminium hydroxide, phosphoric acid
Aluminum or aluminium oxide, oil-Emulsion are (such asOrSaponin and Viamin E solubilisate's
Oil-Emulsion).
Pharmaceutical composition any one of 15. claim 11-14, it is 10 that wherein said pharmaceutical composition comprises dosage3-1010
The D-Glu Auxotrophic mutant of individual antibacterial bacterial isolates alive.
Pharmaceutical composition any one of 16. claim 11-15, wherein said pharmaceutical composition is freeze-dried.
Pharmaceutical composition any one of 17. claim 11-16, wherein said bacterial isolates is selected from the antibacterial consisted of
Species list: Acinetobacter bauamnnii, Acinetobacter baylyi, acinetobacter calcoaceticus, acinetobacter haemolyticus, Qiong Shi are not
Lever bacterium, acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacter pittii, radioprotective are not
Lever bacterium, actinobacillus lignieresii, actinobacillus suis, Aeromonas caviae, Aeromonas hydrophila, Aeromonas veronii
Subsp.Sobria, actinobacillus actinomycetem comitans, Arcobacter butzleri, Arcobacter nitrofigilis, Xie Dian
Afnyloliquefaciens, anthrax bacillus, Bacillus bataviensis, solution fiber bacillus cereus, bacillus cereus, gram Lloyd's's bud
Spore bacillus, Bacillus licheniformis, bacillus megaterium, Bacillus pumilus, bacillus subtilis, bacillus thuringiensis, crisp
Weak bacteroid, Bordetella avium, bordetella branchiseptica, Bordetella pertussis, Bordetella petrii,
Bacillus abortus, brucella melitensis, Brucella suis, Burkholderia cepacia, glanders burkholderia,
Burkholderia multivorans, Burkholderia Pseudomallei, Burkholderia thailandensis, the most curved
Aspergillosis, campylobacter fetus fetus subspecies, campylobacter fetus sexually transmitted disease (STD) subspecies, very thin Campylobacter spp, Campylobacter hominis,
Campylobacter jejuni, rectum Campylobacter spp, Showa Campylobacter spp, Uppsala Campylobacter, citrobacter freundii, kirschner citric acid bar
Bacterium, Clostridium asparagiforme, bacillus botulinus, Bacillus butyricus, clostridium difficile, aerogenesis
Perfringens bacillus, Clostridium saccharobutylicum, Clostridium tetani, Corynebacterium diphtheriae bar
Bacterium, Corynebacterium pseudotuberculosis, clostridium perfringen, enterobacter cloacae, enterococcus faecalis, enterococcus faecalis, erysipelothrix ruhsiopathiae, large intestine bar
Bacterium, actinomyces pseudonecrophorus, fusobacterium nucleatum, adjacent particles chain bacterium, severe foster particle chain bacterium, horse reproductive tract haemophilus, influenza are bloodthirsty
Bacillus, haemophilus parainfluenzae, haemophilus paragallinarum, haemophilus parasuis, pleuropneumonia haemophilus, Haemophilus somnus,
Helicobacter pylori, Klebsiella oxytoca, Klebsiella Pneumoniae, Oak Ridge legionella, legionella pneumophilia, hyperbolic hook end spiral
Body, leptospira illini, leptospria interrogans, Listeria monoeytogenes, capitate lysine bacillus cereus, spherical
Lysine bacillus cereus, cattle catarrhalis, morganella morganii, mycobacterium abscessus, mycobacterium africanum, Mycobacterium avium, cattle are divided
Branch bacillus, Mycobacterium leprae, mycobacterium tuberculosis, gonococcus, Neisseria meningitidis, pasteurella multocida,
Plesiomonas shigelloides, propionibacterium acnes, Proteus hanseri, proteus mirabilis, Pseudomonas aeruginosa, hog cholera
Salmonella, Salmonella enteritidis intestinal subspecies, Salmonella enteritidis, salmonella paratyphi, salmonella typhi, general city sand thunder
Bacterium, shigella boydii, Shigella dysenteriae, shigella flexneri, A Erlaite staphylococcus, staphylococcus aureus, head Fructus Vitis viniferae
Coccus, Staphylococcus caprae, Staphylococcus carnosus, staphylococcus epidermidis, Staphylococcus equorum, staphylococcus haemolyticus, people's Fructus Vitis viniferae ball
Bacterium, road Deng's staphylococcus, Staphylococcus pasteuri, Staphylococcus pettenkoferi, false middle type staphylococcus, corruption
Raw staphylococcus, Staphylococcus simiae, imitate staphylococcus, staphylococcus warneri, stenotrophomonas maltophilia,
Streptococcus agalactiae, streptococcus dysgalactiae, streptococcus dysgalactiae are like horse subspecies, streptococcus equi, streptococcus pneumoniae, micrococcus scarlatinae, breast
Room streptococcus, Streptococcus zooepidermicus, Taylorella asinigenitalis, horse reproductive tract Taylor
Salmonella, treponema carateum, spirochaeta cuniculi, Treponema hyodisenteriae, Tyreponema pallidum,
Treponema suis, Veillonella atypica, different Veillonella, veillonella parvula, rat Veillonella, vibrio cholera,
Vibrio parahaemolytious, Vibrio vulnificans, Yersinia enterocolitica, Yersinia pestis and pseudoconcretion yersinia genus
Bacterium.
The pharmaceutical composition of 18. claim 17, the list of wherein said bacterial isolates choosing freely following species composition: Bao Man is not
Lever bacterium, Pseudomonas aeruginosa and staphylococcus aureus.
The pharmaceutical composition of 19. claim 18, wherein said bacterial isolates is the bacterial isolates of Acinetobacter bauamnnii, its name
For Acinetobacter bauamnnii Delta0380/Delta3398, and by Fundaci ó n Profesor Novoa Santos according to cloth
Da Peisi treaty is deposited in Spain's Type Tissue Collection with strain number 8588 on April 14th, 2014.
The pharmaceutical composition of 20. claim 18, wherein said bacterial isolates is the bacterial isolates of Pseudomonas aeruginosa, its name
For Pseudomonas aeruginosa DeltaPA4662, and by Fundaci ó n Profesor Novoa Santos according to Budapest bar
About it is deposited in Spain's Type Tissue Collection with strain number 8589 on April 14th, 2014.
The pharmaceutical composition of 21. claim 18, wherein said bacterial isolates is the bacterial isolates of staphylococcus aureus, its life
Entitled ..., and by Fundaci ó n Profesor Novoa Santos according to budapest treaty with strain number ...
It is deposited in Spain's Type Tissue Collection on April 14th, 2014.
22. 1 kinds of D-Glu Auxotrophic mutants bacterial isolateses alive, wherein said bacterial isolates is Pseudomonas aeruginosa
Bacterial isolates, its named Pseudomonas aeruginosa DeltaPA4662, and by Fundaci ó n Profesor Novoa
Santos is deposited in Spain American Type Culture Collection with strain number 8589 on April 14th, 2014 according to budapest treaty
Center.
23. 1 kinds of D-Glu Auxotrophic mutants bacterial isolateses alive, wherein said bacterial isolates is Staphylococcus aureus
The bacterial isolates of bacterium, its named 132deltamurI/deltadat, and by Fundaci ó n Profesor Novoa
Santos is deposited in Spain American Type Culture Collection with strain number 8587 on June 11st, 2014 according to budapest treaty
Center.
24. bacterial isolateses defined as any one of claim 22 or 23, as medicine, particularly vaccine.
Any one of 25. claim 1-21 pharmaceutical composition or claim 22 or 23 any one of D-Glu
Auxotrophic mutant bacterial isolates alive is thin for the Auxotrophic mutant of described compositions for animal and/or people
The prophylactic treatment (before infection) of the infection of the wild-type form of bacteria strain and/or therapeutic treatment are (after infection or infect
After the clinical manifestation of caused disease) method.
26. antibody or it is selected from Fab, F (ab') 2, the fragment of Fv, scFv, di-scFv and sdAB, it is capable of identify that D-Glu
Auxotrophic mutant bacterial isolates alive, wherein said antibody or its fragment are suitable for animal and/or people for described group
The prophylactic treatment (before infection) of the infection of the wild-type form of the Auxotrophic mutant bacterial isolates of compound and/or control
The property treated treatment (after the clinical manifestation of the disease after infection or caused by infection).
27. antibody or it is selected from Fab, F (ab') 2, the fragment of Fv, scFv, di-scFv and sdAB, it lacks with D-Glu nutrition
Obtaining after swaged mutant bacterial isolates immunising mammals alive and maybe can obtain, wherein said antibody or its fragment are suitable for
Animal and/or people are for infection preventative of the wild-type form of the Auxotrophic mutant bacterial isolates of described compositions
Treatment (before infection) and/or therapeutic treatment (after the clinical manifestation of the disease after infection or caused by infection).
28. 1 kinds of pharmaceutical compositions, preferably a kind of vaccine, it comprises the antibody any one of claim 26 or 27 or its fragment
And pharmaceutically acceptable carrier or diluent and the adjuvant being optionally present, wherein said pharmaceutical composition be suitable for animal and/or
People (infects it for the preventative of infection of the wild-type form of the Auxotrophic mutant bacterial isolates of described compositions
Before) and/or therapeutic treatment (after the clinical manifestation of the disease after infection or caused by infection).
The pharmaceutical composition of 29. claim 28, wherein said pharmaceutical composition is vaccine, and the most described vaccine
Comprise adjuvant.
Antibody any one of 30. claim 26 or 27 or its fragment, be used for treating, especially for passive immunity.
Antibody any one of the pharmaceutical composition of 31. claim 28 or claim 26 or 27 or its fragment, be used for moving
Thing and/or people control for the preventative of infection of the wild-type form of the Auxotrophic mutant bacterial isolates of described compositions
Treat (before infection) and/or therapeutic treatment (after the clinical manifestation of the disease after infection or caused by infection) method.
In pharmaceutical composition any one of 32. claim 11-21 or claim 28 or claim 22 or 23 arbitrary
Antibody any one of the D-Glu Auxotrophic mutant of item bacterial isolates alive or claim 26 or 27 or its sheet
Section, for animal and/or people for the infection of the wild-type form of the Auxotrophic mutant bacterial isolates of described compositions
Prophylactic treatment (before infection) and/or therapeutic treatment (after infection or infect caused by disease clinical manifestation it
Method afterwards), and wherein said compositions, bacterial isolates or antibody or its fragment are via intranasal, Intradermal, subcutaneous, oral, logical
Cross aerosol, intramuscular, wing lattice and eye drop to be administered.
33. 1 kinds of antibody comprised any one of claim 26 or 27 or the test kit of its fragment or device.
34. 1 kinds for by the test kits of infection of immunoassay detection bacterial origin or device, it comprises:
The i first antibody being referred to as " capture antibody " that () defines as any one of claim 26 or 27, wherein said first resists
Body is preferably attached to solid support;
(ii) being referred to as second traget antibody of " detection antibody ", it identifies the region in addition to the region of first antibody identification, its
Described in second antibody comprise labelling, described labelling can be fluorescence, shiner or enzyme;
(iii) show the reagent of the affinity to second antibody, described reagent be coupled to combine to the first member;And
(iv) combine to the second member, it is coupled to fluorescence, shiner or enzyme, and wherein said combination is to being selected from: hapten
And antibody;Antigen and antibody;Biotin and avidin;Biotin and Succ-PEG-DSPE;Biotin analog
And avidin;Biotin analog and Succ-PEG-DSPE;Sugar and agglutinin;Enzyme and cofactor;Nucleic acid or core
Acid-like substance and complementary nucleic acid or nucleic acid analog.
Test kit any one of 35. claim 33 or 34 or the purposes of device, for qualitative and/or quantitatively determine from the food in one's mouth
The biological sample of breast animal, particularly suspects the bacterial species in the blood plasma of the mammal suffering from bacterial disease or Bacteria
Strain.
The method of 36. 1 kinds of In vitro culture D-Glu auxotrophic bacterium bacterial strains, described method includes utilizing 0.00001-
The D-Glu concentration of 120mM.
The method of 37. claim 36, the concentration range of wherein said D-Glu is 0.01-50mM.
The method of 38. claim 37, the concentration range of wherein said D-Glu is 10-20mM.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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ESP201331504 | 2013-10-11 | ||
ES201331504 | 2013-10-11 | ||
EP14382153 | 2014-04-25 | ||
EP14382153.6 | 2014-04-25 | ||
PCT/EP2014/071926 WO2015052350A2 (en) | 2013-10-11 | 2014-10-13 | Live attenuated vaccines |
Publications (1)
Publication Number | Publication Date |
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CN105899228A true CN105899228A (en) | 2016-08-24 |
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ID=51691060
Family Applications (1)
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CN201480067569.8A Pending CN105899228A (en) | 2013-10-11 | 2014-10-13 | Live attenuated vaccines |
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US (2) | US10517939B2 (en) |
EP (3) | EP3613430A2 (en) |
JP (2) | JP2016535766A (en) |
CN (1) | CN105899228A (en) |
AU (1) | AU2014333706A1 (en) |
BR (1) | BR112016007919A2 (en) |
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CN108359630A (en) * | 2018-04-12 | 2018-08-03 | 南开大学 | The construction method of one plant of attenuated pseudomonas aeruginosa and its application in protein transfection |
CN108441457A (en) * | 2018-06-20 | 2018-08-24 | 中国农业科学院特产研究所 | A kind of preparation method of mink source D types Pseudomonas aeruginosa and its application, its inactivated vaccine and the inactivated vaccine |
CN108660229A (en) * | 2018-05-25 | 2018-10-16 | 上海交通大学医学院附属上海儿童医学中心 | Evaluate pyemic biomarker |
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CN114324889A (en) * | 2021-12-01 | 2022-04-12 | 清华大学 | Vaccine quality control method, vaccine quality control reagent and application thereof |
WO2023098711A1 (en) * | 2021-12-01 | 2023-06-08 | 清华大学 | Vaccine quality control method, vaccine quality control reagent, and use thereof |
CN117187146A (en) * | 2023-11-07 | 2023-12-08 | 中国兽医药品监察所 | Drug-resistance-free rough brucellosis protection strain and application thereof |
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Also Published As
Publication number | Publication date |
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WO2015052350A2 (en) | 2015-04-16 |
WO2015052350A3 (en) | 2015-07-16 |
EP3613430A2 (en) | 2020-02-26 |
BR112016007919A2 (en) | 2017-12-05 |
CA2926984A1 (en) | 2015-04-16 |
US20160235834A1 (en) | 2016-08-18 |
EP3054970A2 (en) | 2016-08-17 |
JP2016535766A (en) | 2016-11-17 |
US10517939B2 (en) | 2019-12-31 |
EP3054970B1 (en) | 2019-04-17 |
EP3552621A2 (en) | 2019-10-16 |
JP2020019801A (en) | 2020-02-06 |
US11197922B2 (en) | 2021-12-14 |
US20200206337A1 (en) | 2020-07-02 |
AU2014333706A1 (en) | 2016-05-26 |
EP3552621A3 (en) | 2020-01-08 |
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